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.

Cardiac autonomic denervation and functional response to neurotoxins during acute experimental Chagas' disease in rats.

Severe cardiac autonomic denervation occurs in the acute Chagas' disease in rats. The present study aims at verifying whether this denervation was accompanied by impairment of heart function. Scorpionic (Tityus serrulatus) crude venom was used for neurotransmitter release in isolated hearts (Langendorff's preparation). In control hearts, the venom induced significant bradycardia followed by tachycardia. In infected animals, despite the severe (sympathetic) or moderate (parasympathetic) cardiac denervation, the venom provoked similar bradycardia but the tachycardia was higher. The hearts of infected animals beat at significantly lower rate. Atropine prevented this lower rate. Our results demonstrated sympathetic dysfunction during the acute phase of Trypanosoma cruzi infection in rats, the parasympathetic function being spared.

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.

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.

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.

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.

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.

Effects of crude venom, tityustoxin and toxin Ts-gamma from Tityus serrulatus scorpion on secretion and structure of the rat submandibular gland.

Changes in the rat submandibular glands after intravenous injections of Tityus serrulatus scorpion venom, tityustoxin or toxin Ts-gamma were studied histologically and morphometrically. The acini and the granular convoluted tubules presented the most prominent changes. The following variables were measured: (a) relative volume occupied by the glandular structures; (b) diameter of the granular convoluted tubules and thickness of their epithelium; (c) diameter of the acini. The cytoplasm of the acinar cells was extensively occupied by large confluent vacuoles and had a reduced number of secretory granules after intravenous injections of venom or toxins. The morphological changes caused by toxin Ts-gamma were greater than those evoked by tityustoxin or crude venom injections. In spite of the changes in acinar cells, acinar diameter showed no significant alterations after venom or toxin injections. Reduction of diameter and depletion of the cytoplasmic secretory granules were observed in the granular convoluted tubules 2 h after intravenous injections of crude venom, or after 1 h with tityustoxin or toxin Ts-gamma. The intravenous injection of crude venom did not induce any visible change in the granular convoluted tubules after 1 h. These structural changes could explain the concomitant intense sialagogue effect elicited by crude venom, tityustoxin and toxin Ts-gamma. The sialagogue effect induced by toxin Ts-gamma was larger than those induced by crude venom or tityustoXin.

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.

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.

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%).

Lung oedema induced by Tityus serrulatus scorpion venom in the rat.

The effects of drugs were investigated on the induction of acute lung oedema by scorpion Tityus serrulatus venom in male Wistar rats (200-230 g) anaesthetized with sodium pentobarbital (40 mg/kg, i.p.). Intravenous (i.v.) injection of scorpion venom (0.5 mg/kg) into 12 rats induced arterial hypertension and severe lung oedema, whereas i.v. injection of scorpion venom into 16 rats previously injected with commercial heparin induced arterial hypertension, but only a slight lung oedema. It is suggested that the inhibitory effect of commercial heparin on the genesis of lung oedema may be due to a decrease in vascular permeability in the lungs. Previous i.v. injection of aprotinin did not prevent the arterial hypertension and the lung oedema induced by scorpion venom. Previous injections of platelet-activating factor antagonists (BN-52021 and WEB-2170) or of an inhibitor of lipo- and cyclooxygenase (Nordihydroguaiaretic acid) did not prevent the arterial hypertension induced by scorpion venom, but decreased the magnitude of the lung oedema elicited by the venom. Previous injections of inhibitors of 5-lipoxygenase (MK-886) or cyclooxygenase (aspirin or indomethacin) significantly decreased the magnitude of the lung oedema induced by scorpion venom. It is concluded that the release of vascular permeability factors, such as platelet-activating factors, leukotrienes, and prostaglandins may play a role in the induction of acute lung oedema by scorpion venom in 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).

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

The effects of toxin Ts-gamma and tityustoxin purified from Tityus serrulatus scorpion venom were investigated on isolated rat atria. Rat atria were placed in an organ bath containing Krebs-Ringer solution, 30 degrees C, pH 7.4, and bubbled with a gas mixture of 95% O2 and 5% CO2. The atrial rate and contractile force were simultaneously recorded. Addition of toxin Ts-gamma to the bath (0.14 microM) evoked an initial reduction of both atrial rate and contractile force, followed by a small increase in force and a decrease in rate, and finally a long reduction of rate and force. Addition of an identical dose of Ts-gamma 30 or 60 min later did not evoke any effect. Addition of tityustoxin to the bath (0.14 microM) induced an increase of atrial rate and force. Addition of an identical dose of tityustoxin 30 min later evoked similar effects. The negative chronotropic and inotropic effects induced by Ts-gamma were abolished by tetrodotoxin (TTX, 1 microM) or atropine (1.5 microM), whereas the positive effects observed in the presence of atropine were prevented by TTX (1 microM) or alprenolol (10 microM). The negative chronotropic effect of 0.14 microM tityustoxin was only observed in the presence of physostigmine (0.3 microM). This negative effect was abolished by TTX (1 microM) or atropine (1.5 microM). The positive inotropic effect of tityustoxin was decreased by TTX (1 microM and 10 microM), but was totally prevented by guanethidine (10 microM) or alprenolol (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Effects of anesthetics on the incidence and duration of reperfusion arrhythmias in isolated rat heart.

The present study was carried out to evaluate the effect of anesthetics on reperfusion arrhythmias. Male Wistar rats (200-300 g) were injected ip with heparin (200 IU), followed by anesthesia with thiopental (40 mg/kg), pentobarbital (30 mg/kg), urethane (1.2 g/kg), either, or halothane and sacrificed by decapitation. The isolated heart (5 to 8 per group) was perfused with Locke solution by the Langendorff method and the left coronary artery was ligated for 10 min. The incidence of reperfusion arrhythmias (100%) was similar in hearts of control and previously anesthetized rats, but the duration of the arrhythmias was significantly increased by anesthesia (5-fold with thiopental, 15-fold with pentobarbital, ether and halothane, and 30-fold with urethane). In hearts taken from unanesthetized rats and perfused with Locke solution containing anesthetics (5-7 per group), the duration of reperfusion arrhythmias decreased with thiopental (0.23 +/- 0.15 min), did not change with pentobarbital (1.14 +/- 0.26 min) and increased with urethane (16.10 +/- 5.60 min). Our results show that anesthetics alter the duration of reperfusion arrhythmias in the isolated rat heart.

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 induced by saliva of the aquatic hemipteran Belostoma anurum on the isolated guinea-pig heart.

1. The effects of the saliva of the aquatic hemipteran Belostoma anurum were investigated on the isolated guinea-pig heart, perfused with Locke solution by the Langendorff's method. 2. The electrocardiogram (bipolar lead), the contractile force (g) and coronary flow (ml/min) were simultaneously recorded. 3. The total protein present in the saliva of each belostomatid was 11.17 +/- 2.39 micrograms (N = 10), and SDS-PAGE of the saliva showed a complex protein composition (N = 10). 4. Bolus injections of 0.2 ml saliva obtained from 10 belostomatids elicited complex effects which were divided into two phases (N = 8): an initial phase characterized by sinus bradycardia and/or A-V block associated with a decrease in contractile force and an increase in coronary flow followed by a decrease and a second phase, 0.5-5 min after saliva injection, characterized by a progressive increase in resting tension (ventricular contracture). 5. Since pretreatment of the preparation with atropine or verapamil did not prevent the initial and the late effects (N = 12), we conclude that these effects are not explained either by a muscarinic effect or by a stimulation of calcium channels. 6. Injection of saliva previously mixed with heparin (1000 IU/ml, N = 7), evoked the first but not the second phase (ventricular contracture). 7. Injection of saliva from two belostomatids into the isolated hearts of guinea pigs, not previously treated with heparin, elicited dramatic effects, such as ventricular contracture (N = 6). 8. We suggest that the formation of an acid-base complex (heparin-saliva) would prevent, in part, the toxic effects of the saliva on the heart.