Age effects on the pharmacokinetics of tityustoxin from Tityus serrulatus scorpion venom in rats.

The pharmacokinetics of scorpion venom and its toxins has been investigated in experimental models using adult animals, although, severe scorpion accidents are associated more frequently with children. We compared the effect of age on the pharmacokinetics of tityustoxin, one of the most active principles of Tityus serrulatus venom, in young male/female rats (21-22 days old, N=5-8) and in adult male rats (150-160 days old, N=5-8). Tityustoxin (6 microg) labeled with 99mTechnetium was administered subcutaneously to young and adult rats. The plasma concentration vs time data were subjected to non-compartmental pharmacokinetic analysis to obtain estimates of various pharmacokinetic parameters such as total body clearance (CL/F), distribution volume (Vd/F), area under the curve (AUC), and mean residence time. The data were analyzed with and without considering body weight. The data without correction for body weight showed a higher Cmax (62.30 +/- 7.07 vs 12.71 +/- 2.11 ng/ml, P<0.05) and AUC (296.49 +/- 21.09 vs 55.96 +/- 5.41 ng h(-1) ml(-1), P<0.05) and lower Tmax (0.64 +/- 0.19 vs 2.44 +/- 0.49 h, P<0.05) in young rats. Furthermore, Vd/F (0.15 vs 0.42 l/kg) and CL/F (0.02 +/- 0.001 vs 0.11 +/- 0.01 l h(-1) kg(-1), P<0.05) were lower in young rats. However, when the data were reanalyzed taking body weight into consideration, the Cmax (40.43 +/- 3.25 vs 78.21 +/- 11.23 ng kg(-1) ml(-1), P<0.05) and AUC (182.27 +/- 11.74 vs 344.62 +/- 32.11 ng h(-1) ml(-1), P<0.05) were lower in young rats. The clearance (0.03 +/- 0.002 vs 0.02 +/- 0.002 l h(-1) kg(-1), P<0.05) and Vd/F (0.210 vs 0.067 l/kg) were higher in young rats. The raw data (not adjusted for body weight) strongly suggest that age plays a pivotal role in the disposition of tityustoxin. Furthermore, our results also indicate that the differences in the severity of symptoms observed in children and adults after scorpion envenomation can be explained in part by differences in the pharmacokinetics of the toxin.

Age effects on the pharmacokinetics of tityustoxin from Tityus serrulatus scorpion venom in rats

The pharmacokinetics of scorpion venom and its toxins has been investigated in experimental models using adult animals, although, severe scorpion accidents are associated more frequently with children. We compared the effect of age on the pharmacokinetics of tityustoxin, one of the most active principles of Tityus serrulatus venom, in young male/female rats (21-22 days old, N = 5-8) and in adult male rats (150-160 days old, N = 5-8). Tityustoxin (6 µg) labeled with 99mTechnetium was administered subcutaneously to young and adult rats. The plasma concentration vs time data were subjected to non-compartmental pharmacokinetic analysis to obtain estimates of various pharmacokinetic parameters such as total body clearance (CL/F), distribution volume (Vd/F), area under the curve (AUC), and mean residence time. The data were analyzed with and without considering body weight. The data without correction for body weight showed a higher Cmax (62.30 ± 7.07 vs 12.71 ± 2.11 ng/ml, P < 0.05) and AUC (296.49 ± 21.09 vs 55.96 ± 5.41 ng h-1 ml-1, P < 0.05) and lower Tmax (0.64 ± 0.19 vs 2.44 ± 0.49 h, P < 0.05) in young rats. Furthermore, Vd/F (0.15 vs 0.42 l/kg) and CL/F (0.02 ± 0.001 vs 0.11 ± 0.01 l h-1 kg-1, P < 0.05) were lower in young rats. However, when the data were reanalyzed taking body weight into consideration, the Cmax (40.43 ± 3.25 vs 78.21 ± 11.23 ng kg-1 ml-1, P < 0.05) and AUC (182.27 ± 11.74 vs 344.62 ± 32.11 ng h-1 ml-1, P < 0.05) were lower in young rats. The clearance (0.03 ± 0.002 vs 0.02 ± 0.002 l h-1 kg-1, P < 0.05) and Vd/F (0.210 vs 0.067 l/kg) were higher in young rats. The raw data (not adjusted for body weight) strongly suggest that age plays a pivotal role in the disposition of tityustoxin. Furthermore, our results also indicate that the differences in the severity of symptoms observed in children and adults after scorpion envenomation can be explained in part by differences in the pharmacokinetics of the toxin.

Technetium-99m labeling of tityustoxin and venom from the scorpion Tityus serrulatus.

The tityustoxin, the most toxic fraction from scorpion Tityus serrulatus venom, has been used as a tool in several neurochemical and neuropharmacological studies. Biological activities of labeled and unlabeled tityustoxin and venom were compared. The samples were labeled in the presence of stannous chloride and sodium borohydride with a yield of 60-70% for the venom and 75-85% for tityustoxin and then chromatographed in Sephadex G-10. Biological activities of tityustoxin and venom were preserved after labeling.

Release of gamma-[(3)H]aminobutyric acid in rat brain cortical slices by alpha-scorpion toxin.

In this paper, the effect of the alpha-scorpion toxin tityustoxin (TsTX) in the release of gamma-[(3)H]aminobutyric acid ([(3)H]GABA) from rat brain cortical slices is described. The TsTX-stimulatory effect on the release of [(3)H]GABA was dependent on incubation time and TsTX concentration, having an EC(50) of 0.33 microM. Tetrodotoxin (TTX) completely inhibited the TsTX action on [(3)H]GABA release. The scorpion toxin effect was calcium-dependent and involves P/Q calcium channels. beta-Alanine also induces the release of [(3)H]GABA that was not inhibited by TTX but was additive in the presence of TsTX. The data suggest a neuronal origin for the release of [(3)H]GABA by TsTX.

Beta-scorpion toxin induces the release of gamma-[3 H]aminobutyric acid in rat brain slices.

The effect of the beta-scorpion toxin, TiTX gamma on the release of [3H]GABA from rat brain cortical slices is described. The stimulatory effect of TiTX gamma on the release of [3H]GABA was dependent on incubation time and TiTX gamma concentration with an EC50 of 0.19 microM. The scorpion toxin effect was calcium dependent and was completely inhibited by tetrodotoxin. beta-Alanine also induced the release of [3H]GABA and this effect was not inhibited by tetrodotoxin but was additive in the presence of TiTX gamma. The data suggest a neuronal origin for the release of [3H]GABA by TiTX gamma.

Spider neurotoxins block the beta scorpion toxin-induced calcium uptake in rat brain cortical synaptosomes.

In this paper we describe the effects of the beta scorpion toxin Tityus gamma (TiTX gamma) and spider neurotoxins Tx3-3 and Tx3-4 in the (45)Ca(2+) uptake in synaptosomes. The TiTX gamma-stimulatory effect on (45)Ca(2+) uptake in synaptosomes was inhibited omega-Conotoxin MVIIC (omega-CgTX MVIIC) (0.1 microM) and omega-Agatoxin IVA (0.1 microM) by 70% and 41%, respectively. omega-CgTX MVIIC (1.0 microM) almost completely blocked the TiTX gamma-induced (45)Ca(2+) uptake in synaptosomes. Verapamil (1.0 microM) and omega-Conotoxin GVIA (0.1 microM) had no effect in the scorpion toxin-induced (45)Ca(2+) influx. The spider neurotoxins Tx3-3 and Tx3-4 inhibited the TiTX gamma-induced calcium uptake with an IC(50) of 10.0 and 30.0 nM, respectively. It is suggested that spider neurotoxins Tx3-3 and Tx3-4 blocking effect in the TiTX gamma-induced calcium uptake involves P/Q-type calcium channels.

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.

Tityustoxin effect on nerve compound action potentials requires extracellular sodium.

Previous studies have demonstrated that Li(+) ions can substitute for Na(+) in a variety of functional systems. Using the single sucrose-gap recording technique, we measured the nerve compound action potential to study the effects of tityustoxin (an alpha-scorpion toxin that selectively inhibits fast Na(+) channel inactivation) upon removal of extracellular Na(+). Our results suggest that tityustoxin requires the presence of extracellular Na(+) to produce its typical pharmacological effect on Na(+) channel inactivation kinetics, but not to bind to its site.

Malnutrition sequela on the drug metabolizing enzymes in male Holtzman rats.

The effect of food restriction on the specific activities of the drug metabolizing enzymes (DME) system was studied in Holtzman male rats by comparing DME activities in 90-day-old control rats fed ad libitum (CO), rats fed 40% restricted food (RF) from the gestation period to the day of sacrifice, and recovered rats (rRF) fed 40% restricted food from period of gestation to 45 days of age and then fed ad libitum until the day of sacrifice. In liver, total cytochrome P450 (CYP) of the RF and rRF groups was higher by approximately 50% and 28%, respectively, than in CO rats. Specific activities of individual CYP monooxygenases (MO) such as CYP2B [7-methoxycoumarin demethylase (MOCD)], CYP1A [aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin deethylase (EORD)], and CYP2E [nitrosodimethylamine demethylase (NDMAd)] were 31, 61, 43, and 56% in RF and 16, 36, 26, and 32% in rRF groups, respectively, more than the CO values. Conjugases such as UDP- glucuronosyltransferases with substrates 3-OH benzo(a)pyrene (UGT1) and 4-hydroxybiphenyl (UGT2) and glutathione S-transferase (GST) with substrate 1-chloro-2,4-dinitrobenzene were higher by 72, 69, and 33% in RF and 28, 38, and 24% in rRF groups, respectively. MO activities (MOCD and EORD) were significantly higher in lung, kidney, and intestine: MOCD by 82, 48, and 45% in RF and 40, 25, and 22% in rRF, respectively; and EORD by 84, 77, and 67% in RF and 40, 33, and 28% in rRF, respectively. However, activity of conjugases (UGT1 and GST) were significantly lower (approximately 35-45%) in RF and rRF rats (approximately 20-30%) than in the CO group in above mentioned extrahepatic tissues. These studies indicate that undernourishment during the period of gestation, weanling, and growth and development of microsomal enzymes produces a sequela of events on the DME in hepatic and extrahepatic tissues that cannot return to the control values even when fed ad libitum.

Alpha- and beta-scorpion toxins evoke glutamate release from rat cortical synaptosomes with different effects on [Na+]i and [Ca2+]i.

Scorpion toxins have long been used as tools in the investigation of neurotransmitter release mechanisms. We have used rat cortical synaptosomes to study the effects of a beta-type scorpion toxin (TiTX-gamma) on the release of glutamate and on the concentrations of free sodium and calcium ions inside the synaptosomes. The effects are compared with those of an alpha-type scorpion toxin (TsTX), on which there have been more studies. TsTX increased overall internal sodium and calcium ion concentrations and glutamate release in an incremental, dose dependent manner. TiTX-gamma similarly evoked glutamate release in an incremental, dose dependent manner. However, TiTX-gamma caused little increase in the overall internal sodium and calcium ion concentrations at low doses that evoked a significant release of glutamate and a maximal increase in these ions at somewhat higher doses. The results suggest that TiTX-gamma preferentially binds sodium channels close to the active zones for glutamate release and indicates that modifications of the activation or inactivation of the Na+-channel can lead to very different changes in the cytosolic concentrations of free Na+and Ca2+, with consequences for neurotransmission. This provides an interesting perspective concerning modulation of neurotransmitter release via pharmacological manipulation of Na+-channel properties, that may lead to a better comprehension of its physiological and pathological roles.

A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis.

1. The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. 2. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. 3. Using omega-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by omega-agatoxin IVA, an antagonist of P/Q calcium channels. Omega-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. 4. It is concluded that Tx3-3 potently inhibits omega-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

Tityustoxin-induced release of ATP from rat brain cortical synaptosomes.

Tityustoxin, a scorpion toxin that alters the Na+ channel activity, induces release of ATP from rat brain cortical synaptosomes. The effect of tityustoxin is dependent on its concentration and incubation time. Continuously or cumulative release of ATP evoked by tityustoxin was calcium-dependent and interestingly only partially inhibited by tetrodotoxin. We suggest that tityustoxin mainly releases ATP from the vesicular pool but other pools may also be involved.

Different effects of reducing agents on omega-conotoxin GVIA inhibition of [3H]-acetylcholine release from rat cortical slices and guinea-pig myenteric plexus.

1. The effect of reducing reagents on omega-conotoxin GVIA (omega-CgTX) inhibition of the release of [3H]-acetylcholine ([3H]-ACh) induced by tityustoxin, K+ 50 mM and electrical stimulation was investigated in rat brain cortical slices. 2. In cortical slices the inhibition of tityustoxin or electrically-stimulated [3H]-ACh release by omega-CgTX was dramatically increased by reducing reagents ascorbate or beta-mercaptoethanol. Dehydroascorbic acid did not substitute for ascorbate. 3. Depolarization induced by K+ 50 mM caused [3H]-ACh release from cortical slices which was not inhibited by omega-CgTX, even in the presence of ascorbate. 4. In the guinea-pig myenteric plexus, omega-CgTX inhibition of the tityustoxin induced release of [3H]-ACh was independent of ascorbate. 5. It is suggested that N-type-like calcium channels in guinea-pigs myenteric plexus may have pharmacological/biochemical diversity from similar channels of rat cerebral cortex.

The effect of PhTx3 on the release of 3H-acetylcholine induced by tityustoxin and potassium in brain cortical slices and myenteric plexus.

The venom of the Brazilian spider Phoneutria nigriventer possesses several neurotoxic polypeptidic fractions. Previous work has established that one of the toxic components, PhTx3, inhibited Ca(2+)-dependent glutamate release and the increase in cytosolic free Ca2+ in response to membrane depolarization. In the present work, we investigated the effect of PhTx3 on the release of acetylcholine (ACh) from brain and peripheral neurons. PhTx3 decreased the release of [3H]-ACh induced by tityustoxin and KCl in brain cortical slices and myenteric plexus. The inhibitory effect of myenteric plexus had the same magnitude as that obtained in the absence of extracellular Ca2+. However, in brain PhTx3 was less efficient at decreasing the evoked release of ACh. These experiments suggest that the target of PhTx3 is coupled to the process of release of ACh in brain and autonomic nervous system.

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)

Differential effects of calcium channel antagonists on tityustoxin and ouabain-induced release of [3H]acetylcholine from brain cortical slices.

In this paper, the effect of calcium channel blockers on acetylcholine release induced by tityustoxin and ouabain in rat brain cortical slices is described. Cadmium, a non-specific blocker of calcium channels, inhibited the release of ACh induced by tityustoxin. L-type calcium channel blockers (verapamil, nifedipine and diltiazen) had no effect on the release of ACh induced by tityustoxin. The release of ACh was also unaffected by nickel, a T-type calcium channel blocker, and the conotoxins GVIA and MVIIC, blockers of N and Q-type calcium channels. Agatoxin IVA, a specific blocker of the P-type calcium channel, inhibited the release of ACh induced by tityustoxin by 50%. The spontaneous release of ACh as well as ouabain-induced release of ACh was unaffected by any of the calcium channel blockers studied. It is concluded that ACh release induced by tityustoxin is mediated by Ca2+ influx via P-type calcium channels.

Tityustoxin-mediated Na+ influx is more efficient than KCl depolarisation in promoting Ca(2+)-dependent glutamate release from synaptosomes.

The scorpion venom toxin, tityustoxin (TsTX), causes rapid, dose-dependent increases in intracellular free Ca2+ ([Ca2+]i) and glutamate release in rat cerebrocortical synaptosomes. These effects are completely abolished by the Na+ channel blocker tetrodotoxin (TTX). The increase in [Ca2+]i is completely dependent on extracellular Ca2+ but the increased glutamate release has both Ca(2+)-dependent and -independent components. Comparison of the effects of TsTX with those of depolarising concentrations of KCl reveals that TsTX is more effective, both in raising [Ca2+]i and promoting Ca(2+)-dependent and -independent glutamate release. These data suggest that the Ca(2+)-dependent glutamate release caused by TsTX is only partly due to Ca2+ entry through voltage-sensitive Ca2+ channels.

Ageing and acetylcholine release by tityustoxin from brain cortical slices.

The ability of tityustoxin to trigger acetylcholine release was studied in cerebral cortical slices from 2, 12 and 23-month-old rats. The effect of tityustoxin to induce the release of acetylcholine was reduced by 60% in senescent animals. This result suggests that ageing reduces the sensitivity of cerebral cortical nerve terminals to tityustoxin.

Effect of lanthanum ions on the release of acetylcholine induced by tityustoxin, K+ and ouabain from myenteric plexus and brain cortical slices.

Exposure of myenteric plexus longitudinal muscles to 2 mM LaCl3 increased the rate of spontaneous [3H]-ACh release. In brain cortical slices La3+ did not affect the spontaneous release of ACh. The release of ACh induced by tityustoxin, ouabain or K+ 50 mM in brain cortex and myenteric longitudinal muscles slices was inhibited by La3+ treatment. It is suggested that tityustoxin, ouabain and K+ release ACh from the same pool that is inhibited by La3+ pretreatment.