Effect of atrial natriuretic peptide on the release of beta-endorphin from rat hypothalamo-neurohypophysial complex

The aim of this study was to determine whether atrial natriuretic peptide (ANP) alters beta-endorphin (beta-END) secretion from rat intermediate pituitary and whether this effect is a direct action on the intermediate pituitary or an indirect one mediated by hypothalamic factor(s). We studied the release of beta-END from rat neuro-intermediate lobes of the pituitary (NIL) and from the hypothalamo-neurohypophysial complex (HNC), which consists of the hypothalamus, pituitary stalk, intermediate and posterior lobes of the pituitary, by means of an in vitro perifusion system. NIL and HNC were prepared from male Wistar rats and individually perifused for 30 min with perifusion medium followed by 20 min perifusion with medium containing alpha-rat ANP and/or dopamine (DA). Samples of perifusion medium were collected every 5 min and subjected to RIA for beta-END. The basal release of beta-END from NIL was 180% of that from HNC (p less than 0.01), which provides further support for the presence of hypothalamic factors that inhibit beta-END release from the intermediate pituitary. The perifusion of HNC with ANP at 10(-7) and 10(-6) M increased the beta-END concentration by 25 and 50%, respectively (p less than 0.01). In contrast, ANP (10(-8) to 10(-6) M) had no effect on beta-END release from NIL. The inhibitory effect of DA (10(6) M) on beta-END release from NIL and HNC (51% and 50% of the basal release, respectively, p less than 0.01) was confirmed. However, this inhibitory effect was not reversed by ANP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scorpion neurotoxin: Mode of action on neuromuscular junctions and synaptosomes

Electrophysiological analysis of the effects of scorpion toxin I, one of the neurotoxins from the venom of the scorpion Androctonus australis Hector, upon crayfish neuromuscular junctions has shown that the toxin strongly associates with the nerve terminal to stimulate release of neurotransmitters.The biochemical approach has shown that the binding of scorpion toxin I to rat brain synaptosomes is accompanied by a decrease in their capacity to accumulate γ-aminobutyric acid. The main effect of the toxin is to stimulate neurotransmitter release. The apparent dissociation constant of the toxin-receptor complex is 0.1–0.2 μM at 22 °C. The rate of dissociation is so slow that complex formation seems to be quasi-irreversible. The “quasi-irreversibility” has also been observed in electrophysiological experiments with the crayfish neuromuscular junction. Tetrodotoxin prevents scorpion toxin I action if it is incubated with synaptosomes or with crayfish neuromuscular junctions before scorpion toxin I application. Tetrodotoxin does not reverse scorpion toxin action if it is added to the preparation after scorpion toxin I. Prevention of scorpion toxin action by tetrodotoxin permits measurements of binding characteristics of this toxin to synaptosomes. The dissociation constant of the tetrodotoxin-receptor complex is 2.2 nM at 22 °C. No cooperativity is observed in the binding. Because of its high affinity for synaptosomes (and the “quasi-irreversibility” of the binding), scorpion toxin I appears to be a potentially excellent tool for further studies of the molecular mechanism of neurotransmitter secretion.     

Evidence that free radical generation occurs during scorpion envenomation

Although it is well established that symptomatology, morbidity and death following scorpion envenomation are due to increases in neurotransmitter release secondary to toxins binding to voltage-sensitive sodium channels, the mechanism by which venom action is involved in damaging heart, liver, lungs and kidneys remains unclear. We hypothesized that scorpion toxins could induce the generation of high levels of free radicals responsible for membrane damage in organs targeted by venom action. We have investigated lipid peroxidation in different organs, through the evaluation of thiobarbituric acid reactive substances (TBARS), after experimental envenomation of rats by toxic fractions of Androctonus australis Hector venom. We have shown that scorpion toxins cause considerable lipid peroxidation in most vital organs. We also evaluated the protective effects of antioxidants in mice injected with lethal doses of toxins. Among the drugs tested, N-acetylcysteine (NAC) was effective in protecting the mice when injected prior to toxin application. However, the free radical scavenging properties of NAC seem less implicated in these protective effects than its ability to increase the fluidity of bronchial secretions. We therefore conclude that free radical generation only plays a minor role in the toxicity of scorpion venom.     

Preliminary X-ray diffraction studies on a scorpion neurotoxin: Toxin II of Androctonus australis Hector

The most toxic scorpion neurotoxin (toxin II), isolated from the North African scorpion Androctonus australis Hector, has been crystallized. The space group is (C2 with one molecule of protein in the asymmetric unit. The cell parameters are a = 51.9 Å, b = 33.3 Å, c = 40.4 Å, β = 113 °. The crystals are very stable under radiation.     

Aging and photoperiod affect the daily rhythm pattern of atrial natriuretic peptide in the rat atrium

This study investigates the release characteristics of atrial natriuretic peptide (ANP) from young (10 weeks) and old (22 months) rat atrium. Levels of ANP release from samples of atrium were studied by organ perifusion. Rats were exposed to light:dark (LD) cycles of 12:12 or 18:6 and sacrificed at different zeitgeber time (ZT) points: ZT0, ZT6, ZT8, ZT12, ZT16, and ZT19 for LD 12:12 or ZT0, ZT9, ZT16, ZT18, ZT20, and ZT 21.5 for LD 18:6. The heart was collected, and the right atrium was removed, weighed, and perifused with Krebs-bicarbonate buffer for 100 min, including a period of 50 min for stabilization of secretion rate. ANP concentrations released by atrium did not differ between the two age groups either under LD 12:12 or under LD 18:6, except at the light:dark transition under LD 12:12 conditions where ANP levels were significantly (P < 0.05) lower in young compared to old rats. ANP exhibited daily variations in concentrations under LD 12:12, with a peak during the beginning of photophase (ZT0) in young rats and a peak at the beginning of scotophase (ZT12) in old animals. These variations were strongly modified under LD 18:6, where the pattern of the release exhibited a peak during the light phase at ZT16 in both young and old rats. This strongly suggests that the atrial ANP rhythm is dependent on the environmental light:dark cycle. Moreover, the total ANP levels released by atria in old rats were significantly increased under LD 18:6 compared to standard LD 12:12. This observation strongly suggests that old animals are more sensitive to a photoperiodic change. In conclusion, our results show that ANP concentrations in the rat atrium exhibit daily variations which are significantly affected by the daylength (photoperiod) change in aged rats.     

Construction and functional evaluation of a single-chain antibody fragment that neutralizes toxin AahI from the venom of the scorpion Androctonus australis hector.

9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom. It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals. Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single-chain antibody variable fragment molecule (scFv). This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L-agarose beads yielding 1-2 mg.L(-1) of bacterial culture. scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI. The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range. Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes. Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1. Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.     

Low-affinity gamma-aminobutyric acid transport in rat brain

The low-affinity (Km = 100-200 microM) gamma-aminobutyric acid (GABA) transporter from membrane vesicles from rat brain has been characterized and found to be in many aspects similar to the well-known sodium- and chloride-coupled high-affinity gamma-aminobutyric acid transporter (Km = 2-4 microM). Influx by this system is sodium and chloride dependent and stimulated by an interior negative membrane potential. Steady-state levels obtained by both systems are lowered by the sodium channel openers veratridine and aconitine. However, while the channel blocker tetrodotoxin fully reverses this inhibition with the high-affinity system, this is not the case for its low-affinity counterpart. Furthermore, the toxin from the scorpion Androctonus australis Hector inhibited high-affinity transport only. Efflux of gamma-aminobutyric acid taken up by the high-affinity system displayed a Km of about 100 microM. Exchange catalyzed by the low-affinity system was observed in the absence of external sodium and chloride. Furthermore, both activities copurified in the fractionation procedure developed to purify the high-affinity transporter. All these observations are consistent with the idea that both activities are manifestations of only one gamma-aminobutyric acid transporter. The high-affinity binding site represents the extracellular and the low-affinity site the cytosolic aspect of the transporter. In addition, it was found that right-side-out synaptosomes also contain a low-affinity GABA transporter. This apparently represents a different transport protein.     

Crystallization and preliminary X-ray study of AaH IT2, and insect-specific toxin from the scorpion Androctonus australis Hector

An insect-specific toxin from the venom of the scorpion Androctonus australis Hector has been crystallized. The crystals are orthorhombic, space groups P2(1)2(1)2(1), with cell dimensions a = 66.4 A, b = 52.5 A and c = 36.1 A. Calculations based on the unit cell volume and toxin molecular mass suggest that there are two molecules in the asymmetric unit.     

Use of antibodies specific to defined regions of scorpion alpha-toxin to study its interaction with its receptor site on the sodium channel

Five antibody populations selected by immunoaffinity chromatography for their specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the 125I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to their antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only alpha-helix region (residues 23-32) and a beta-turn structure (residues 32-35) are accessible to their respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.     

Wheat germ in?vitro translation to produce one of the most toxic sodium channel specific toxins

Envenoming following scorpion sting is a common emergency in many parts of the world. During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim causing serious medical problems. The exploration of toxin structure-function relationship would benefit from the generation of soluble recombinant scorpion toxins in Escherichia coli. We developed an in vitro wheat germ translation system for the expression of the highly toxic Aah (Androctonus australis hector)II protein that requires the proper formation of four disulphide bonds. Soluble, recombinant N-terminal GST (glutathione S-transferase)-tagged AahII toxin is obtained in this in vitro translation system. After proteolytic removal of the GST-tag, purified rAahII (recombinant AahII) toxin, which contains two extra amino acids at its N terminal relative to the native AahII, is highly toxic after i.c.v. (intracerebroventricular) injection in Swiss mice. An LD₅₀ (median lethal dose)-value of 10 ng (or 1.33 pmol), close to that of the native toxin (LD₅₀ of 3 ng) indicates that the wheat germ in vitro translation system produces properly folded and biological active rAahII. In addition, NbAahII10 (Androctonus australis hector nanobody 10), a camel single domain antibody fragment, raised against the native AahII toxin, recognizes its cognate conformational epitope on the recombinant toxin and neutralizes the toxicity of purified rAahII upon injection in mice.     

Inhibition of atrial natriuretic peptide secretion by forskolin in noncontracting cultured atrial myocytes

Secretory rates for immunoreactive atrial natriuretic peptide (ANP) by 7 - 8 day-old primary cultures of atrial myocytes from adult rats (with myocyte contraction inhibited by tetrodotoxin (TTX)) were (a) constant for at least two hours, and (b) significantly slowed by forskolin (1, 5, and 25 microM), dibutyryl cyclic adenosine monophosphate (1 mM), or isobutylmethylxanthine (100 microM). The substantial rates of ANP secretion which persisted in cells rendered noncontracting either by inhibiting Ca2+ influx via reduction of external [Ca2+] to less than 10(-7) M or by inhibiting sarcoplasmic reticulum Ca2+ release with 100 microM ryanodine were significantly slowed by 25 microM forskolin, but forskolin sensitivity was lost by cells exposed simultaneously to external Ca2+ concentration of less than 10(-7) M and 100 microM ryanodine. Quiescent myocytes whose ANP secretory rate was depressed by forskolin remained responsive to secretory stimulation by phorbol ester.     

Effect of sinus node on spontaneous release of natriuretic peptide in isolated atria

Experiments were conducted to examine the release of atrial natriuretic peptide (ANP) in an isolated atrium in the presence and absence of sinus node tissue. The first series of experiments were conducted with the aid of a metabolic chamber to examine the spontaneous release of ANP by the right atrium with and without the sinus node region. The left atrium was also studied. The right atrium with the sinus node, quiescent right atrium without the sinus node, and the left atrium were incubated at 35 degrees C in 10 mL of oxygenated Tyrode's solution. After 40 min of equilibration, the incubation medium was removed at 10-min intervals for the determination of immunoreactive ANP concentration. The right atria with the sinus node released the highest amount of ANP into the incubation medium (32.2 +/- 2.7 pg.min-1.mg-1), compared with quiescent right atria (20.9 +/- 3.7 pg.min-1.mg-1). The left atria released the least amount of ANP into the incubation medium (9.9 +/- 1.5 pg.min-1.mg-1) when compared with the quiescent right atria and the right atria. In the second series of experiments, the right atrium was divided into the sinus node region and the quiescent right atrium, and these tissues were studied in paired fashion with a modified Langendorff preparation. The right atrium without the sinus node and sinus node region were perfused with Tyrode's solution, equilibrated with 95% O2 and 5% CO2 at 37 degrees C with a constant flow of 0.5 mL/min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and immunological characterization of two peptides which are models for two of the four major antigenic sites of a scorpion toxin

We have synthesized, by the solid phase procedure, then purified and chemically characterized two peptides. They mimic two regions of toxin II of the scorpion Androctonus australis Hector, one around disulfide bridge 12-63 and another at sequence 50-59. Each of these two regions was supposed to include an antigenic site. We have shown that the synthetic replicas of these regions are individually recognized by a part of IgGs raised against native toxin II. This is a strong argument for the involvement of these areas in the antigenicity of the toxin. Furthermore, region-specific IgGs purified by affinity chromatography on the two Sepharose linked peptides were able to bind 125I-labelled toxin II.     

Developmental changes in inotropic actions of neurotoxins observed in ventricular muscle of rat heart.

Developmental changes in functions of myocardial sodium channels were examined from inotropic effects of several neurotoxins in ventricular muscle preparations obtained from prenatal (20-22 day gestation) or adult (3-4 months old) rat hearts. Tetrodotoxin caused a negative inotropic effect in low concentrations and a loss of muscle responsiveness to electrical stimulation in high concentrations in preparations obtained from either prenatal or adult rat heart. The tetrodotoxin concentration that caused a 50% decrease in developed tension was higher in prenatal rats. Anemonia sulcata toxin, Androctonus australis toxin, veratridine, and Centruroides sculpturatus toxin all produced positive inotropic effects in adult rat heart. The effects were largest with A. sulcata and A. australis toxins, intermediate with veratridine, and smallest with C. sculpturatus toxin. Prenatal heart required higher concentrations of either veratridine, or A. sulcata or A. australis toxins to produce comparable positive inotropic effects. With C. sculpturatus toxin, no significant positive inotropic effect was observed in prenatal heart muscle preparations. These results indicate that cardiac sodium channels undergo significant functional changes during development and that negative and positive inotropic effects of neurotoxins resulting from inhibition and enhancement of fast Na+ channels reflect developmental changes in the cardiac sodium channels.     

Tityus serrulatus toxin VII bears pharmacological properties of both beta-toxin and insect toxin from scorpion venoms

Some beta-toxins from the South American scorpion Tityus serrulatus (e.g. Ts VII) are highly toxic both for mouse and fly larva. Radioiodinated Ts VII and the insect toxin from the North African scorpion Androctonus australis Hector (AaH IT) bind to the same site on a house fly head synaptosomal fraction. These results reinforce the hypothesis about the existence of a correlated series of scorpion toxins as previously defined by amino acid compositions and sequences, and immunological and circular dichroism studies, in suggesting that Ts VII constitutes a link which may fill the pharmacological gap existing between beta-toxins and insect toxins such as AaH IT.     

An equilibrium ELISA for the dosage of Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot) scorpion venoms: set up and calibration

Scorpion stings are very frequent in Tunisia; yet a method for evaluating envenoming severity and consequently victim treatment, has never been adequately established nor has its efficiency been properly evaluated. Indeed, a management of envenomed patients requires the optimization of envenoming antivenom immunotherapy. This task requires either, an accurate evaluation of toxicokinetic parameters of scorpion venoms in absence and in presence of antivenom, using animals as models, and the establishment of a quantitative relationship between human blood scorption venom levels, envenoming severities and clinical symptoms. A performant sandwich ELISA was set up and calibrated for measuring scorpion venom levels in human and rabbit sera. This assays was performed with polyclonal F(ab')2 specific to the two North African scorpion (Androctonus australis garzonii; Aag and Buthus occitanus tunetanus: Bot) venoms. It is simple, rapid, very sensitive (detection limit = 0.9 ng/ml) and shows a good linearity for venom concentrations in human sera comprised between 0.5 and 15 ng/ml. The ELISA is also reproducible: the coefficient of variation, determined at different venom concentrations (low: 4 ng/ml; medium: 8 ng/ml and high: 12 ng/ml) prepared in a pool of sera collected from several healthy donors, were lower than 10%. Such an ELISA has been successfully used, either in experimental toxinokinetic and immunotherapeutic studies carried out in rabbits or for the quantification of Aag and Bot venom levels in the serum of human victims stung by these scorpion.     

Augmentation of insulin-stimulated ANP release through tyrosine kinase and PI 3-kinase in diabetic rats

The aim of present study was to define the effects of insulin on atrial dynamics and ANP release and its modification in diabetic rats. An isolated perfused beating atrial model was used from control and diabetic rats. Insulin was perfused with and without an inhibitor for tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Insulin increased the release of ANP and decreased atrial contractility in a dose-dependent manner. During the perfusion of 10(-10)M insulin, the release of ANP abruptly increased within 8min by approximately 40% and then decreased with time despite of continuous perfusion. In terms of increasing the dose of insulin, the time to reach the peak effect became faster and the slope to decrease became slower. In contrast, atrial contractility was gradually decreased with time. These effects were independent upon extracellular glucose. Genistein (10(-5)M) or lavendustin C (10(-5)M), a tyrosine kinase inhibitor, attenuated the release of ANP stimulated by insulin (10(-8)M). Wortmannin (10(-7)M) or LY294002 (10(-5)M), a PI 3-kinase inhibitor, also attenuated insulin-stimulated ANP release. However, both inhibitors for PI 3-kinase and tyrosine kinase did not cause any significant effects on negative inotropism by insulin. Insulin-stimulated ANP release was augmented in streptozotocin-treated rat atria. The density of insulin receptor markedly increased in diabetic hearts. These results suggest that insulin stimulates the release of ANP through PI 3-kinase and tyrosine kinase, and augmentation of insulin-stimulated ANP release in diabetic rat atria may be partly due to an upregulation of insulin receptor.     

Two-dimensional 1H nuclear magnetic resonance study of AaH IT, an anti-insect toxin from the scorpion Androctonus australis Hector. Sequential resonance assignments and folding of the polypeptide chain

Sequence-specific nuclear magnetic resonance assignments for the polypeptide backbone and for most of the amino acid side-chain protons, as well as the general folding of AaH IT, are described. AaH IT is a neurotoxin purified from the venom of the scorpion Androctonus australis Hector and is specifically active on the insect nervous system. The secondary structure and the hydrogen-bonding patterns in the regular secondary structure elements are deduced from nuclear Overhauser effects and the sequence locations of the slowly exchanging amide protons. The backbone folding is determined by distance geometry calculations with the DISMAN program. The regular secondary structure includes two and a half turns of alpha-helix running from residues 21 to 30 and a three-stranded antiparallel beta-sheet including peptides 3-5, 34-38, and 41-46. Two tight turns are present, one connecting the end of the alpha-helix to an external strand of the beta-sheet, i.e., turn 31-34, and another connecting this same strand to the central one, i.e., turn 38-41. These structure elements are very similar to the secondary structure reported in single crystals for either variant 3 from the scorpion Centruroides sculpturatus Ewing (CsE V3) or toxin II from the scorpion A. australis Hector (AaH II). The differences in the specificity of these related proteins, which are able to discriminate between mammalian and insect voltage-dependent sodium channels of excitable tissues, are most probably brought about by the position of the C-terminal peptide with regard to a hydrophobic surface common to all scorpion toxins examined thus far. This surface is made of an aromatic cluster that is surrounded by long hydrophobic side-chain residues, as well as the loops protruding out of it. Thus, the interaction of a given scorpion toxin with its receptor might well be governed by the presence of this solvent-exposed hydrophobic surface, whereas adjacent areas modulate the specificity of the interaction.     

Effects of several sea anemone and scorpion toxins on excitability and ionic currents in the giant axon of the cockroach

The membrane effects of 4 sea anemone and 6 scorpion toxins have been studied under current clamp and voltage clamp conditions. Micromolar concentrations of the purified toxins were applied externally on single giant axons of the american cockroach. Periplaneta americana in a double oil-gap arrangement and the effects on the resting potential, action potential and underlying currents analysed. The 4 sea anemone toxins (Condylactis toxin, Anemonia toxin 2, Anthopleurin toxin A and Parasicyonis toxin) were found to considerably prolong the action potential. This effect is frequency dependent and long plateau spikes (100-500 ms in duration) are consistently seen for frequencies lower than 0.2 Hz. This effect is due to a considerable delay in the turning-off of the sodium current during square membrane depolarizations associated, for large concentrations, with a decrease in the potassium conductance. Toxin effects on the sodium current are not prevented by pretreatment with STX. From the 4 purified toxins extracted from the venom of the scorpion, Androctonus australis Hector, 3 (Mammal toxins 1 and 2 and crustacean toxin) were found to have sea anemone toxin like effects and to induce long duration plateau action potentials. As for sea anemone toxins, this effect is due to a lengthening of the falling phase of the sodium current associated with a small decrease in the potassium conductance. The 4th toxin (insect toxin or ITAaH) depolarizes the membrane and induces repetitive firing of short action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of monoclonal antibodies. Scorpion antitoxins: characterization and molecular mechanisms of neutralization

Two monoclonal antibodies specific for the potent toxin II of the scorpion Androctonus australis Hector have been produced. One of them shows both high affinity binding to the toxin (Kd = 0.8 nM) and in vivo and in vitro neutralizing properties. The mechanism by which the antibody neutralizes toxin binding to its receptor was shown to be of the competitive type, the epitope is overlapping or being close to the receptor binding region of the toxin. Several residues of the toxin clustered in the C-terminal region were shown likely to be part of the discontinuous epitope recognized by the antibody. The positive charge of the NH2-Lys58 seems to play a pivotal role in the binding of the toxin to both the monoclonal antibody and the sodium channel receptor.