Scorpion venom-induced neutrophilia is inhibited by a PAF receptor antagonist in the rat.

A dramatic blood neutrophilia is an important feature of the severe envenoming caused by the Brazilian scorpion Tityus serrulatus and may contribute to the development of lung injury in children. We examined the effects of an intravenous injection of T. serrulatus scorpion venom (TsV) on the total number of leukocytes and neutrophils in the blood of anesthetized rats. Injection of TsV (250 microg/kg) induces a significant leukocytosis 2 and 3 h after its injection, explained by an increase in the number of neutrophils. The release of catecholamines and action on adrenoceptors is responsible for most of the systemic manifestations of TsV. However, pretreatment with the beta-adrenoceptor antagonists metoprolol and propranolol or the alpha1-adrenoceptor antagonist prazosin (0.25 mg/kg) did not prevent TsV-induced neutrophilia. Blood neutrophilia induced by TsV occurred simultaneously with a significant reduction of mature neutrophils in bone marrow. Pretreatment with the platelet-activating factor (PAF) receptor antagonists UK-74505 or WEB-2086 prevented TsV-induced increase in blood neutrophils and reduction in the number of neutrophils in the bone marrow. It is concluded that scorpion venom induces blood neutrophilia in rats, explained by a PAF receptor-dependent mobilization of neutrophils from the bone marrow.

Pharmacological evidence that neuropeptides mediate part of the actions of scorpion venom on the guinea pig ileum.

Severe human scorpion envenoming is characterised by instability of several physiological systems and death. These manifestations are explained by the ability of the venom toxins to activate sodium channels in nerve terminals with the subsequent release of neurotransmitters, specially acetylcholine and noradrenaline. However, there is evidence to suggest that other neurotransmitters are also released. We now have sought evidence for a role of the substance P receptor, the tachykinin NK1 receptor, in mediating part of the contractile actions of Tityus serrulatus venom on the isolated guinea pig ileum. Scorpion venom induced a significant elevation of baseline tension with frequent and periodic superimposed contractions on the elevated baseline. Pretreatment with atropine partially blocked the elevation in baseline and in the number of superimposed contractions. These responses were also partially inhibited by the tachykinin NK1 receptor antagonist, CP96,345 (the dihydrochloride salt of (2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)methyl)-1-az abicycol[2.2.2]octan-3-amine), but not by its inactive enantiomer, CP96,344 (the 2R-3R enantiomer of CP96,345). Pretreatment with the combination of atropine and CP96,345 completely inhibited the effects of the venom. Moreover, pretreatment with the combined drugs abolished the effects of toxin gamma, a toxin purified from the venom. Finally, another tachykinin NK1 receptor antagonist, RP67,580 ((3aR, 7ar)-7,7-diphenyl-2-[1-imino-2-(2-methoxy-phenyl)ethyl]perhydro isoindol-4-one), significantly inhibited the venom-induced contractions. These results demonstrate an important role for NK1 receptors in mediating part of the contractile effects of the venom on guinea pig ileum. The release of neuropeptides may play an important role in the systemic manifestations of severe envenoming.

Effects of tachykinin NK1 or PAF receptor blockade on the lung injury induced by scorpion venom in rats.

In cases of severe human scorpion envenoming, lung injury is a common finding and frequently the cause of death. In the rat, two distinct mechanisms account for oedema following the intravenous injection of the venom -- acute left ventricular failure resulting from a massive release of catecholamines and an increase in pulmonary vascular permeability. In the present work, we investigated the effects of a tachykinin NK1 receptor antagonist (CP96,345, the dihydrochloride salt of (2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)methyl)-1-az abicycol[2.2.2]octan-3-amine) and its 2 R-3 R inactive enantiomer (CP96,344) on the acute lung injury induced by the i.v. injection of Tityus serrulatus venom in rats. Lung injury was assessed by evaluating the extravasation of Evans blue dye in the bronchoalveolar lavage fluid and in the lung of venom-treated and control animals. The effects of the platelet-activating factor (PAF) receptor antagonist WEB2170 (2-methyl-1-phenylimidazol[4,5c]pyridine) were evaluated for comparison. The i.v. injection of the venom induced the extravasation of Evans blue in the bronchoalveolar lavage fluid and into the left lung. Pretreament with the tachykinin NK1 receptor antagonist CP96,345, but not CP96,344, inhibited Evans blue dye extravasation in the bronchoalveolar lavage fluid and in the lung by 96% and 86%, respectively. The PAF receptor antagonist WEB2170 inhibited the increase in vascular permeability in the bronchoalveolar lavage fluid by 60% and had no effect on the extravasation to the lung parenchyma of venom-injected animals. In addition to abrogating lung injury, pretreatment of rats with CP96,345, but not CP96,344 or WEB2170, decreased by 70% the mortality induced by the venom. This is the first study to show the relevance of the tachykinin NK1 receptor in mediating lung injury and mortality in animals injected with the neurotoxic T. serrulatus venom. Blockade of the tachykinin NK1 receptor may represent an important strategy in the treatment of patients with signs of severe envenoming and clearly deserves further studies.

Heparin or a PAF antagonist (BN-52021) prevents the acute pulmonary edema induced by Tityus serrulatus scorpion venom in the rat.

Experiments were performed in pentobarbital-anesthetized rats, to study the mechanism of the acute pulmonary edema induced by Tityus serrulatus scorpion venom. In control rats injection of venom (50 micrograms/100 g, i.v.) induced arterial hypertension and lung edema (lung/body index or LBI equal to 1.01 +/- 0.09). In rats pretreated with heparin (100 IU/100 g 30 min previously) the venom induced similar hypertensive effects, but no edema was detected (LBI = 0.63 +/- 0.06, P > 0.05). Similarly, in rats pretreated with the PAF antagonist BN-52021 (0.5 mg/100 g, i.v., 30 min previously), the venom-induced hypertension was not modified but the acute pulmonary edema was prevented (LBI = 0.67 +/- 0.08, P > 0.05). It is concluded that PAF plays an important role on the genesis of pulmonary edema induced by scorpion venom in the rat. It is suggested that the inhibitory action of heparin could be related to a decrease in the vascular permeability in the lungs.

Effects of a purified scorpion toxin (tityustoxin) on the isolated guinea pig heart.

To study the cardiac effects induced by purified scorpion toxin (tityustoxin, TsTX), without circulatory and respiratory influences, isolated guinea pig hearts were used. Single injections of 10, 20 or 30 micrograms TsTX induced positive inotropic and chronotropic effects in the majority of the experiments. The inotropic effects were dose-dependent. Simultaneous recording of the electrical activity of the heart showed sinus tachycardia, T wave inversion and ST segment deviations. Sinus bradycardia and/or A-V block were recorded in some experiments, simultaneously with the positive inotropic effects. After these initial events, the larger dose of TsTX elicited periodic changes of heart rate (in 70% of the experiments) which were explained by wandering pacemakers. During these periodic changes in heart rate, the electrocardiogram showed sinus bradycardia, idioventricular rhythm, complete or partial A-V block, sinus tachycardia, sinus arrest and junctional rhythm. The coronary flow varied inversely with the inotropism and the cardiac rate, but the changes in flow were related mainly to the inotropism. The effects of TsTX on the electrocardiogram, inotropism and coronary flow spontaneously disappeared 15-20 min after toxin injection. The arrhythmias induced by TsTX were blocked either by propranolol or by atropine and are assumed to be due to the release of catecholamines and acetylcholine from postganglionic nerve fibers in the heart.

Immunotherapy for scorpion envenoming in Brazil.

Using the ELISA we have shown that in rats subcutaneously injected with Tityus serrulatus scorpion venom there is a fast absorption rate, a fast and high distribution of venom to tissues, a great affinity of the venom for the tissues and a slow elimination half-life. Because of these experimental data, i.v. immunotherapy should be given to patients stung by scorpions as soon as possible after hospital admission. The severity of scorpion envenoming is related to plasma venom concentration (ELISA). The high levels of plasma scorpion venom antigens (ELISA) were cleared 1 h after the infusion of antivenom (5-30 ml of Fab2 fragment) and high concentrations of circulating antivenom persisted for at least 24 h, confirming the efficacy of immunotherapy to neutralise circulating venom. Some symptoms (e.g. local pain and vomiting) decreased 1 h after the starting of immunotherapy, whereas the other symptoms disappeared from 12-48 h later. Using our tripartite approach of treating scorpion envenoming (symptomatic measures, support of vital functions and serotherapy), the mortality rate was very low (0.28%).

Approaches to the treatment of scorpion envenoming.

A total of 3866 patients stung by Tityus serrulatus scorpion was admitted to Hospital João XXIII, in Belo Horizonte, Brazil, over a 16-year period (an average of 241 cases per year). Of these, 73% were adults and 27% were children aged less than 14 years. The moderate or benign cases were treated with symptomatic measures and/or i.v. antivenom, whereas 168 severely envenomed children were treated in the Intensive Care Unit. Lung oedema was unilateral in several cases, with the presence of air bronchograms and a peripheral distribution, suggesting that a noncardiogenic factor is also involved in the genesis of lung oedema. The treatment consisted of symptomatic measures, support of vital functions and i.v. antivenom. The mortality was 1% among children and 0.28% for the total number of patients.

Effect of purified scorpion toxin (tityustoxin) on the pancreatic secretion of the rat.

Intravenous injection, in anesthetized rats, of a single dose of purified scorpion toxin (tityustoxin, TsTX), obtained from the venom of the Brazilian scorpion Tityus serrulatus, causes a striking increase in flow rate, protein content, kallikrein and amylase activities of the pancreatic juice. The flow rate and protein content of the juice remain significantly higher than in control rats, for at least one hour, whereas the kallikrein activity returns to control values 30 min after tityustoxin injection. Sub-diaphragmatic bilateral vagotomy does not prevent the pancreatic secretion induced by tityustoxin; moreover, vagotomy potentiates the flow rate and kallikrein secretion produced by the toxin. Pre-treatment of the rats with atropine blocks the pancreatic secretion evoked by tityustoxin. It is suggested that the pancreatic secretion induced by tityustoxin is due to actions of acetylcholine, released from postganglionic nerve fibers, on muscarinic receptors. The mechanism by which vagotomy potentiates the pancreatic secretion evoked by tityustoxin is under investigation.

Effect of Tityus serrulatus scorpion toxin on serum gastrin levels in anaesthetized rat.

Scorpion toxin induces gastric secretion of acid and pepsin in rats. These effects seem to be mediated by the release of acetylcholine and histamine. However, the role of gastrin in the scorpion-toxin-induced gastric secretion is unknown. We describe the effects of the T1 fraction purified from Tityus serrulatus scorpion venom on serum and on antral tissue gastrin levels in anaesthetized rats. Gastrin levels in serum and in the antral mucosa were measured before and at intervals 5, 15, 30, 60, 90 up to 120 min after the intravenous injection of saline or the T1 fraction of scorpion venom (0.25 mg/kg) into anaesthetized rats. Antral G-cells were submitted to immunocytochemistry and electron microscopy. The data on gastrin were correlated with the gastric juice volume, and the acid and pepsin output increases induced by toxin. Scorpion toxin induced a significant increase in volume, acid output and pepsin output of gastric juice and gastrin serum levels 15-60 min after injection. Simultaneous measurements of antral gastrin levels did not show significant effects. The number of dense, intermediate and empty granules per microm(2) in the cytoplasm of antral G-cells was not significantly changed 60 min after saline or toxin injection. Scorpion toxin significantly increased serum gastrin; levels in rats.

Effects of crotoxin on the isolated guinea pig heart.

The effects of crotoxin, isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus, were investigated on isolated guinea pig hearts, perfused with Locke solution, by the Langendorff method. The cardiac beats and the electrocardiogram were simultaneously registered and the creatine kinase (CK) activity of the perfusate measured. Crotoxin was infused (4.5 x 10(-8) M and 2.3 x 10(-7) M) into the heart during 90 min, and induced a remarkable decrease in the contractile force, without a significant reduction of heart rate, increased the P-R interval and displaced the S-T segment. The activity of CK only increased in the late phases of the experiments, when the force of contraction was below 25% of the control value. Arrhythmias were uncommon and no alterations of QRS duration or Q-Tc interval were observed. The reduction of the contractile force and the increase in CK activity were completely prevented by bovine serum albumin, whereas lanatoside C did not interfere with the toxin action. A bolus injection of crotoxin (11 +/- 2 nmoles) also induced a decrease of contractile force without reduction of heart rate. This decrease of force was partially prevented by indomethacin, but not by atropine. It is suggested that the reduction of contractile force evoked by crotoxin is due probably to release of free fatty acids and lysophospholipids (initial effect) and to a cellular lesion (late effect).

Effect of scorpion toxin from Tityus serrulatus on the contraction of the isolated rat uterus.

Scorpion toxin T1 from Tityus serrulatus was tested for its effects on the isolated rat uterus preparation. T1 (5 micrograms/ml) caused a contraction of the uterus, which was potentiated by neostigmine (1.64 x 10(-6) M) and abolished by atropine (1.4 x 10(-7) M). After addition of neostigmine to the bath, we noted a higher amplitude of the toxin-induced contractions, and the appearance of repetitive rhythmic contractions. The scorpion toxin-induced contraction was not prevented by previous addition to the bath of hexamethonium or bradykinin, 5-HT and angiotensin II antagonists. The uterine contraction was prevented by previous addition to the bath of either tetrodotoxin (5 x 10(-8) M) or lidocaine (4.2 x 10(-5) M). These data seem to indicate that scorpion toxin-induced rat uterus contractions are due to actions on post-ganglionic autonomic nerve endings, with acetylcholine release and stimulation of muscarinic receptors.

Evidence for a role of mast cells in the lung edema induced by Tityus serrulatus venom in rats.

In the most severe cases of human poisoning by Tityus serrulatus, pulmonary edema is a frequent finding and can be the cause of death. Mast cells can release a range of mediators known to be involved in the development of lung edema following T. serrulatus venom injection. The present work was designed to investigate whether mast cells participated in the acute lung injury induced by T. serrulatus scorpion venom and could, thus, be an intermediate between neuropeptide release and activation of the inflammatory cascade. To this end, mast cells were depleted using compound 48/80. Pulmonary edema, as assessed by the levels of extravasation of Evans blue dye in the bronchoalveolar lavage and in the left lung, was completely inhibited in compound 48/80-treated animals. Moreover, the number of animals surviving 60min after injection of venom rose from 20 to 60%. Our results demonstrate an important role for mast cells in the development of lung injury and lethality following the intravenous administration of T. serrulatus venom.

Effects of toxin Ts-gamma, purified from Tityus serrulatus scorpion venom, on the isolated rat atria.

By using a pair of silver/silver-chloride electrodes it was possible to record, simultaneously, the atrial electrogram and the atrial contractile force of rat atria, in an organ bath, containing Krebs-Ringer solution (30 degrees C, pH 7.4, bubbled with 95% O2 and 5% CO2). Addition of toxin Ts-gamma, purified from Tityus serrulatus scorpion venom, into the bath (1 microgram/ml), evoked complex effects characterized by an initial reduction of both rate and contractile force, followed by increase of force and reduction of rate and finally by reduction of both rate and force. The increase of contractile force was prevented by metoprolol and is, therefore, adrenergic in nature. The reduction of rate was concomitant with changes in the atrial electrogram in which a positive P wave was replaced by a diphasic P wave, while the positive Ta wave was depressed. Experiments with tetrodotoxin, atropine and physostigmine indicate that these effects are due to the release of acetylcholine from vagal endings.

Cardiovascular and respiratory effects induced by a purified scorpion toxin (tityustoxin) in unanesthetized rats.

Intravenous injection of a purified scorpion toxin (tityustoxin) into unanesthetized rats induced acute pulmonary edema, which was directly related to the dose and the time of intoxication. To study the cardiovascular and respiratory effects evoked by an edematogenic dose of the toxin (1 mg/kg), the following parameters were recorded in unanesthetized rats: systolic, diastolic and mean arterial pressure; central venous pressure; electrocardiogram; respiratory movements. The toxin induced acute systolic and diastolic hypertension, bradycardia and bradypnea. During a 1 hr period, the systolic, diastolic and mean arterial pressure fell progressively to control values, whereas the central venous pressure did not change significantly. The cardiac and respiratory rates remained lower than the control values during a 1 hr period. Several changes in the respiratory pattern were recorded, such as gasping, prolonged and labored expiration, ataxic rhythm and noisy inspiration with the mouth open. These respiratory changes were explained, in part, by the presence of edema in the lungs and froth in the trachea. From a group of 24 rats, 6 died 18-30 min after tityustoxin injection. The cause of death was apnea. The female rats were more susceptible to pulmonary edema and death than the male rats.

Effects of purified scorpion toxin (tityustoxin) on gastric secretion in the rat.

An i.v. bolus injection of a purified scorpion toxin (tityustoxin, TsTX) in urethane anesthetized rats induced a dramatic increase in volume, acid and pepsin output of gastric juice and a significant decrease in its pH. The maximal stimulatory effects of TsTX on gastric secretion were obtained with a dose of 0.25 mg/kg acting for 60 min. Hexamethonium did not prevent the gastric secretion evoked by TsTX, whereas atropine or cimetidine abolished partially or totally the toxin effects. Acute bilateral cervical or abdominal vagotomy did not prevent the effects of TsTX on gastric secretion, but chronic abdominal vagotomy abolished the toxin effects. Chronic antrectomy diminished the effect of TsTX on gastric secretion. In the pylorus-ligated group of rats, the gastric secretion evoked by TsTX was not different from that observed in the pylorus-intact group. It is concluded that the changes in gastric volume, acid output, pH and pepsin output induced by TsTX in the rat are due to the release of chemical mediators from postganglionic autonomic nerve fibers which would act through muscarinic and H2-receptors stimulation.

Mechanisms underlying the structural alterations of the rat submandibular gland induced by Tityus serrulatus tityustoxin.

As the mechanisms underlying the structural changes induced in rat submandibular glands by Tityus serrulatus tityustoxin have not been reported, the present study was undertaken to investigate the participation of adrenergic and muscarinic cholinergic receptors in these alterations. Most of the stimulatory effects of the toxin are observed in the secretory cells of the acini and granular convoluted tubules (GCT). We evaluated the ability of the toxin to induce morphological changes in acinar and GCT cells after adrenoreceptor and cholino receptor blockage. The influence of tityustoxin-induced adrenal discharge on the acinar and GCT cells was also investigated after bilateral adrenalectomy. We show that the intense cytoplasmic vacuolation of the acinar cells induced by tityustoxin was prevented by prazosin (alpha(1) adenoreceptor blockade) and atropine (muscarinic cholinoreceptor blockade). The decrease of GCT cell granules following tityustoxin injection was completely blocked by prasozin and partially by propranolol. These results indicate that acinar vacuolation, degranulation of GCT cells, reduction of GCT diameter and height of its epithelium depends on tityustoxin induced adrenergic and cholinergic mechanisms. In contrast, tityustoxin induced acinar cell degranulation was not modified by atropine, prasozin or propranolol (beta(1)-beta(2) adenoreceptor blockade). Thus, acinar degranulation seems to be due to a direct action of tityustoxin on of the rat submandibular glands. The degranulation of the GCT cells and the acinar vacuolation was also prevented by bilateral adrenalectomy, suggesting that these effects are mostly due to catecholamines released from the adrenal glands.

Evidence for a direct action of Tityus serrulatus scorpion venom on the cardiac muscle.

The ability of toxins to activate the cardiovascular system plays an important role in the morbidity and lethality of the Tityus serrulatus scorpion envenoming. Most of the actions of the scorpion toxins are indirect and due to the release of adrenergic and cholinergic neurotransmitters. Accordingly, treatment following envenoming is targeted towards inhibition of adrenergic and cholinergic receptors. Here, we have sought evidence for a direct action of T. serrulatus venom on the isolated rat heart (Langendorff's method). We show that the bradycardia induced by T. serrulatus venom was completely blocked by atropine, a muscarinic receptor antagonist. Similarly, the increase in heart rate that follows the venom-induced bradycardia was totally inhibited by a beta(1)-adrenoceptor antagonist or by chemical sympathetic denervation with 6-hydroxydopamine. In contrast to these findings, the venom-induced increase in contractile force was not modified by beta(1)-adrenoceptor blockade or by chemical sympathetic denervation. The results clearly demonstrate that the chronotropic effects of T. serrulatus are dependent on neurotransmitter release, but the inotropic effects are not. The neurotransmitter-independent increase in contractility seems to be a direct action of the venom on cardiomyocytes. We suggest that this direct effect on cardiac fibers may play a role in the development of cardiac arrhythmias and contractility defects following envenoming with T. serrulatus scorpion.

Acute gastric mucosal injury induced by toxins from Tityus serrulatus scorpion venom: a novel experimental model in the rat.

The effect of a partially purified fraction (T1) and toxin gamma purified from Tityus serrulatus scorpion venom, on gastric mucosa were investigated in anesthetized rats. The animals were injected i.v. with the T1 fraction (37.5 micrograms/100 g) or with saline and 60 min later were sacrificed and the stomachs resected. The gastric juice was measured and stereoscopic examination of the stomachs made. In animals injected with the T1 fraction there was an increase in volume, acidity and pepsin output of rat stomach. The T1 fraction also induced acute gastric injuries in the glandular mucosa, consisting of circular or linear ulcers, and punctiform lesions. Intravenous injection of 20 micrograms/100 g of a pure toxin obtained from Tityus serrulatus scorpion venom (toxin gamma) also induced similar lesions in the rat stomach. Our data indicate that the injection of T1 fraction or toxin gamma are good models to induce acute gastric ulcers in a short period of time in anesthetized rats.

Pharmacokinetics of Tityus serrulatus scorpion venom determined by enzyme-linked immunosorbent assay in the rat.

Experiments were performed in two groups of anaesthetized rats to study the genesis of pulmonary oedema and to determine the pharmacokinetic parameters following a subcutaneous (s.c.) injection of Tityus serrulatus scorpion venom. In group I, the rats were anaesthetized with pentobarbital (4 mg/100 g, i.p.); the s.c. injection of scorpion venom at the dose of 50 micrograms/100 g did not induce arterial hypertension, but unilateral pulmonary oedema was observed in three of six rats. The injection of a higher dose of venom (200 micrograms/100 g, N = 6) induced arterial hypertension and bilateral (N = 3) or unilateral (N = 1) pulmonary oedema. These data indicate that it is possible to evoke unilateral pulmonary oedema without previous arterial hypertension induced by the venom. For the study of pharmacokinetic parameters a second group of six rats was anaesthetized with urethane (140 mg/ 100 g, i.p.) and the venom injected at a dose of 200 micrograms/100 g, s.c. The plasma concentrations of venom were determined by enzyme-linked immunosorbent assay, at times 0, 5, 30, 60, 180, 360, and 720 min after venom injection. A biphasic curve was obtained with an ascending phase followed by a descending phase. The maximum plasma scorpion venom concentration was reached at 60 min. The pharmacokinetic parameters showed a fast absorption rate (Ka = 0.058 min-1), a fast and high distribution of venom to tissues (t1/2 alpha = 31.50 min and Vdarea = 6800.47 ml.kg-1, respectively), a great affinity of the venom for the tissues (KCT = 0.056 min-1 and KTC = 0.002 min-1) and a slow elimination half-life (t1/2 beta = 173.25 min).

Acid-base balance following Tityus serrulatus scorpion envenoming in anaesthetized rats.

In the present work the pH and arterial blood gases were measured in fasted and fed male albino rats, weighing 297 +/- 13 g, anaesthetized with urethane (1.4 g/kg, i.p.) before and after injection of T1 fraction from Titys serrulatus scorpion venom, during 60 min. Arterial blood samples were collected at 0, 5, 15, 30 and 60 min for pH, pCO2, pO2, bicarbonate and base-excess analysis. The data showed that the scorpion toxin induced a continuous drop in the blood pH along the time. Hypercapnia and hypoxemia peaking at 30 min and followed by a recovery towards normal values at 60 min were also observed. A pronounced decrease in the blood bicarbonate levels at 60 min and negative base-excess values along with time were evident at 60 min. The comparisons between fasted and fed animals have shown that in the last group the effects of scorpion toxin on the arterial blood gases were less pronounced. We conclude that T1 fraction of Tityus serrulatus scorpion venom induces in anaesthetized rats an acute respiratory acidosis followed by metabolic acidosis.