Gyroxin and its biological activity: effects on CNS basement membranes and endothelium and protease-activated receptors.

Gyroxin is a glycoprotein isolated from rattlesnake venom, with known thrombin-like serine protease properties and behavioral action in the CNS. The mechanism of the latter has eluded experimenters for three decades. In this paper about the in vitro chick retina we demonstrate an excitotoxic CNS action of Gyroxin by observing retinal Intrinsic Optical Signals (IOS). These show sudden dynamic changes in the intact tissue due to gyroxin action. The very fast kinetics of this response precludes deep tissue penetration by the protein, a mechanism of tissue response described here for the first time. At nanomolar concentrations, Gyroxin alters profoundly the optical profiles of retinal spreading depression waves (RSDs), suggesting modulation of ionic transport and metabolism. This effect is reversible in contrast with the acute cell lysis induced with gyroxin pulses at higher concentration. Because there may be more than one target of Gyroxine at the retinal inner limiting membrane, additional biochemical assays were performed to study a possible Na/K-ATPase blockade and PAR receptor activation. We conclude that the Gyroxin interaction with basement membranes of CNS and endothelium triggers conformational phase transitions at basement membranes, with multiple functional consequences.

Gyroxin increases blood-brain barrier permeability to Evans blue dye in mice.

Gyroxin is a serine protease enzyme component of the South American rattlesnake (Crotalus durissus terrificus) venom. This toxin displays several activities, including the induction of blood coagulation (fibrinogenolytic activity), vasodilation and neurotoxicity, resulting in an effect called barrel rotation. The mechanisms involved in this neurotoxic activity are not well known. Because gyroxin is a member of a potentially therapeutic family of enzymes, including thrombin, ancrod, batroxobin, trypsin and kallicrein, the identification of the mechanism of gyroxin's action is extremely important. In this study, gyroxin was isolated from crude venom by affinity and molecular exclusion chromatography. Analysis of the isolated gyroxin via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein band with a molecular weight of approximately 28 kDa, confirming the identity of the molecule. Furthermore, intravenous administration of purified gyroxin (0.25 µg/g of body weight) to mice resulted in symptoms compatible with barrel rotation syndrome, confirming the neurotoxic activity of the toxin. Mice treated with gyroxin showed an increase in the concentration of albumin-Evans blue in brain extracts, indicating an increase in the blood-brain barrier (BBB) permeability. This gyroxin-induced increase in BBB permeability was time-dependent, reaching a peak within 15 min after exposure, similar to the time span in which the neurotoxic syndrome (barrel rotation) occurs. This work provides the first evidence of gyroxin's capacity to temporarily alter the permeability of the BBB.

Cloning of serine protease cDNAs from Crotalus durissus terrificus venom gland and expression of a functional Gyroxin homologue in COS-7 cells.

Gyroxin is one of main serine proteases of Crotalus durissus terrificus venom, representing about 2% of the protein content in the crude venom. It is a 33 kDa glycoprotein with 3.8% by weight of sugar moiety. This toxin induces hemotoxicity in mice and a neurological condition called barrel rotation syndrome. In the present work, we report the molecular cloning of five new nucleotide sequences from a cDNA library of the venom glands of a single specimen of C. d. terrificus. These sequences have been analyzed in silico with respect to their cDNA organization and similarity with other snake venom serine proteases (SVSPs). We also describe a rapid and efficient method for screening vectors for mammalian cell expression, based on the fact that SVSPs are difficult-to-express toxins due to the presence of several disulfide bonds and glycosylation in their structures. Thus, one of the Gyroxin cDNAs was subcloned into pSectag2 HygroA and pED vectors and used to transfect COS-7 cells. Expression of the functional recombinant Gyroxin isoform was achieved with this cell line with esterase activity in the conditioned culture medium, as revealed by immunoblot of secreted protein and standard anti-crotalic serum from Butantan Institute.

Biodistribution studies of bee venom and spider toxin using radiotracers

The use of radiotracers allows the understanding of the bioavailability process, biodistribution, and kinetics of any molecule labelled with an isotope, which does not alter the molecule's biological properties. In this work, technetium-99m and iodine-125 were chosen as radiotracers for biodistribution studies in mice using bee (Apis mellifera) venom and a toxin (PnTX2-6) from the Brazilian "armed" spider (Phoneutria nigriventer) venom. Incorporated radioactivity was measured in the blood, brain, heart, lung, liver, kidney, adrenal gland, spleen, stomach, testicle, intestine, muscle, and thyroid gland. Results provided the blood kinetic parameter, and different organs distribution rates.(AU)

Blockade of neuronal nitric oxide synthase abolishes the toxic effects of Tx2-5, a lethal Phoneutria nigriventer spider toxin.

The primary goal of this study was to determine whether Tx2-5, a sodium channel selective toxin obtained from the venom of the spider Phoneutria nigriventer, produced penile erection by means of nitric oxide mechanism. Toxin identity was analyzed by MALDI-TOF, ES-MS and N-terminal amino acid sequencing. Pretreating mice with the non-selective nitric oxide synthase (NOS) inhibitor N(omega)-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and the selective neuronal-NOS inhibitor 7-Nitroindazole (7-NI) prior to Tx2-5 i.p. (10 microg/25 g mouse) injection challenged the hypothesis above. Controls were injected with the D-isomer or DMSO or saline. Results demonstrated that L-NAME inhibited penile erections in about half the animals treated, while 7-NI completely abolished this effect. Interestingly 7-NI also abolished all the other symptoms of intoxication induced by Tx2-5, including salivation, respiratory distress and death. Tx2-5 killed all the animals of the control group and no one in the 7-NI-treated group. We conclude that (1) intraperitoneal injections of Tx2-5 induce a toxic syndrome that include penile erection, hypersalivation and death by respiratory distress or pulmonary edema; (2) pretreatment with the non-selective NOS inhibitor L-NAME reduces the penile erection and partially protects from the lethal effects of Tx2-5; (3) pretreatment with the nNOS-selective inhibitor 7-NI completely abolishes all the toxic effects of Tx2-5, including penile erection and death suggesting that nNOS is the major player in this intoxication; (4) toxins from other animals that affect sodium channels in the same way as Tx2-5 and induce similar toxic syndromes may have as a major common target, the activation of nitric oxide synthases.

Gyroxin fails to modify in vitro release of labelled dopamine and acetylcholine from rat and mouse striatal tissue.

Gyroxin fails to modify in vitro release of labelled dopamine and acetylcholine from rat and mouse striatal tissue. Gyroxin is a thrombin-like peptide with amidasic, esterasic and fibrinogenolitic activities, found in the venom of snakes like Lachesis muta muta and Crotalus durissus terrificus. Intravenous injections of small doses of gyroxin induce a typical barrel rotation behaviour that has been thought to be a neurotoxic effect. The aim of this study was to determine whether gyroxin-induced barrel rotation behaviour involves changes in neurotransmitter release. Gyroxin was isolated from crude venoms by gel filtration and affinity chromatography. Its properties were determined by assaying esterasic, amidasic and fibrinogenolitic enzymatic activities and tested for barrel rotation behaviour. Neurotransmitter release tests employed rat and mouse superfused brain striatal chopped tissue preloaded with [(3)H]-dopamine, [(3)H]-acetylcholine or in a double labelling procedure. They were stimulated by 20mM K(+) in control conditions or in the presence of several concentrations of toxins. Crotoxin and crotamine were used as positive controls. Gyroxins failed at modifying both basal and stimulated neurotransmitter releases, suggesting a lack of direct neurotoxic effect. We therefore suggest that gyroxin may not be a neurotoxin but rather, induces this behavioural syndrome by other means possibly related to haemodynamic disturbance. The possible role of vasopressin is discussed.

Striatal dopamine receptor supersensitivity afte long-term haloperiodol treatment of hypophysectomized rats

Dopamine (DA) receptor sensitivity was studied after long-term treatment with haloperidol (0.5 ad 3.0 mg/Kg, ip, single daily dose) or saline in hypophysectomized and infact rats. Haloperidol treatment for seven days produced a 25 to 125% increase in [3H]-spiroperidol binding to strial DA receptors in a dose-dependent fashion. The increase in binding sits (Bmax) was ximilar in both hypophysectomized and intact rats when compared to controls. The present results show that haloperidol treatment

Striatal dopamine receptor supersensitivity after long-term haloperidol treatment of hypophysectomized rats.

Dopamine (DA) receptor sensitivity was studied after long-term treatment with haloperidol (0.5 and 3.0 mg/kg, ip, single daily dose) or saline in hypophysectomized and intact rats. Haloperidol treatment for seven days produced a 25 to 125% increase in [3H]-spiroperidol binding to striatal DA receptors in a dose-dependent fashion. The increase in binding sites (Bmax) was similar in both hypophysectomized and intact rats when compared to controls. The present results show that hypophysectomy does not effect the supersensitivity of striatal DA receptors induced by long-term haloperidol treatment.

Human growth hormone radioiodination using different batches of 125I of various ages.

The reproducibility and the influence of the batch and decay level of Na125I on the radioiodination of human growth hormone (hGH) were examined by a polyacrylamide gel electrophoresis (PAGE) technique. The between-day coefficient of variation (CV) exhibited a value of 11.9% for labelling yields and 14.3% for antibody specific binding. The within-day variation for different shipments and isotope decay-levels was similar to that for the control experiment, carried out under the same conditions, using Na125I from a single lot. The results indicate that the decay-level and batch do not significantly influence either the yields or the immunological properties of the labelled product. The suitability of this PAGE technique as a control test for radioiodinated proteins is established by comparison with gel chromatography on Sephadex G-100.