Effect of the phospholipase A2 inhibitor Varespladib, and its synergism with crotalic antivenom, on the neuromuscular blockade induced by Crotalus durissus terrificus venom (with and without crotamine) in mouse neuromuscular preparations.

The venom of the South American rattlesnake Crotalus durissus terrificus causes an irreversible neuromuscular blockade in isolated preparations due to action of the presynaptically-acting heterodimeric phospholipase A2 (PLA2) crotoxin. Some populations of this subspecies contain, in addition to crotoxin, the toxin crotamine, which acts directly on muscle fibers. In this study we used C. d. terrificus venoms with (crot+) or without (crot-) crotamine to test whether Varespladib, a PLA2 inhibitor, is able to abrogate the neuromuscular blockade induced by these venoms comparatively with crotalic antivenom. Mouse phrenic nerve-diaphragm preparations were exposed to venoms previously incubated with two different concentrations of Varepladib or antivenom, or with a mixture of these two agents, before addition to the bath. In another experimental setting, venoms were initially added to the system, followed by the addition of Varespladib or antivenom 10, 30, or 60 min after venom. At the highest concentrations tested, Varespladib and antivenom inhibited the action of the venom >80% and >70%, respectively. With lower concentrations the inhibition of neuromuscular blockade decreased, but when low doses of the two agents were incubated together with the venom, the inhibitory effect improved, underscoring a synergistic phenomenon. When added after venom, Varespladib was able to halt the progression of the neuromuscular blockade even when added at 60 min. Antivenom exhibited a lower ability to inhibit the toxic effect of the venoms in these conditions. In conclusion, the PLA2 inhibitor Varespladib is highly effective at abrogating the neuromuscular blocking activity of crotamine-positive and crotamine-negative C. d. terrificus venoms and seems to act synergistically with antivenom.

A Highly Polar Phytocomplex Involving Rutin is Responsible for the Neuromuscular Facilitation Caused by Casearia sylvestris (guaçatonga).

BACKGROUND: Of the various biological activities ascribed to extracts from Casearia sylvestris (guaçatonga), its facilitatory activity, i.e., ability to increase skeletal muscle contractile amplitude, has promising therapeutic applications. In this work, we investigated the components responsible for the previously described neurofacilitation caused by C. sylvestris leaves. METHODS: The methanolic fraction of C. sylvestris leaves was initially fractionated by column chromatography and partitioned in a MeOH:H2O gradient. The resulting fractions were analyzed by analytical HPLC and yielded fraction 5:5 (F55) that was subjected to solid phase extraction and preparative HPLC. Of the seven resulting subfractions, only F55-6 caused muscle facilitation. Subfractions F55-6 and F55-7 (similar in composition to F55-6 by TLC analysis, but inactive) were analyzed by 1H-NMR to identify their constituents. RESULTS: This analysis identified a rutin-glycoside phytocomplex that caused neurofacilitation, a property that commercial rutin alone did not exhibit. CONCLUSION: F55-6 apparently caused neurofacilitation by the same mechanism (presynaptic action) as the methanolic fraction since its activity was also inhibited in tetrodotoxin-pretreated preparations.

The Facilitatory Effect of Casearia sylvestris Sw. (guaçatonga) Fractions on the Contractile Activity of Mammalian and Avian Neuromuscular Apparatus.

Many natural products influence neurotransmission and are used clinically. In particular, facilitatory agents can enhance neurotransmission and are potentially useful for treating neuromuscular diseases in which muscular weakness is the major symptom. In this work, we investigated the facilitatory effect of apolar to polar fractions of Casearia sylvestris Sw. (guaçatonga) on contractility in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) neuromuscular preparations exposed to indirect (via the nerve; 3 V stimuli) and direct (30 V stimuli) muscle stimulation in the absence and presence of pharmacological antagonists. Methanolic and ethyl acetate fractions, but not hexane or dichloromethane fractions, exerted a facilitatory effect on PND (indirect stimulation). The methanolic fraction was chosen for further assays to assess the involvement of: 1) presynaptic sites (axons or nerve terminals), 2) postsynaptic sites (cholinergic receptors, sarcolemma or T-tubules), and 3) the synaptic cleft (acetylcholinesterase enzyme). In preparations treated with d-tubocurarine, the methanolic fraction did not cause facilitation in response to direct stimuli; this fraction was also unable to reverse dantrolene-induced blockade (indirect stimulation). In curarized preparations, the methanolic fraction either restored neuromuscular transmission (mimicking the effect of neostigmine) or failed to cause any recovery of neurotransmission. In the presence of 3,4-diaminopyridine (3,4-DAP), the methanolic fraction decreased twitch amplitude, whereas at a high frequency of stimulation (40 Hz) there was an increase in tetanic tension. In BC preparations, the methanolic fraction did not affect contractures to exogenous acetylcholine or potassium chloride. Incubation with atropine showed there was certain modulation by prejunctional nicotinic receptors, whereas treatment with nifedipine showed that the neurofacilitation required the entry of extracellular calcium. Tetrodotoxin did not prevent the facilitatory effect of 3,4-DAP or neostigmine, but antagonized the response to the methanolic fraction. These findings indicate that neuronal sodium channels have an important role in the facilitatory response to the methanolic fraction, with extracellular calcium entry via calcium channels modulating this neurofacilitation. Possible modulation of prejunctional cholinoceptors was not excluded, particularly in view of certain antagonism by the methanolic fraction at muscarinic receptors. Since facilitation by the methanolic fraction involved enhanced acetylcholine release, use of this fraction could be potentially beneficial in neuromuscular diseases and in the reversal of residual paralysis in the post-operative period or after local anaesthesia.

The facilitatory effect of Casearia sylvestris Sw. (guaçatonga) fractions on the contractile activity of mammalian and avian neuromuscular apparatus.

Many natural products influence neurotransmission and are used clinically. In particular, facilitatory agents can enhance neurotransmission and are potentially useful for treating neuromuscular diseases in which muscular weakness is the major symptom. In this work, we investigated the facilitatory effect of apolar to polar fractions of Casearia sylvestris Sw. (guaçatonga) on contractility in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) neuromuscular preparations exposed to indirect (via the nerve; 3 V stimuli) and direct (30 V stimuli) muscle stimulation in the absence and presence of pharmacological antagonists. Methanolic and ethyl acetate fractions, but not hexane or dichloromethane fractions, exerted a facilitatory effect on PND (indirect stimulation). The methanolic fraction was chosen for further assays to assess the involvement of: 1) presynaptic sites (axons or nerve terminals), 2) postsynaptic sites (cholinergic receptors, sarcolemma or T-tubules), and 3) the synaptic cleft (acetylcholinesterase enzyme). In preparations treated with d-tubocurarine, the methanolic fraction did not cause facilitation in response to direct stimuli; this fraction was also unable to reverse dantrolene-induced blockade (indirect stimulation). In curarized preparations, the methanolic fraction either restored neuromuscular transmission (mimicking the effect of neostigmine) or failed to cause any recovery of neurotransmission. In the presence of 3,4-diaminopyridine (3,4-DAP), the methanolic fraction decreased twitch amplitude, whereas at a high frequency of stimulation (40 Hz) there was an increase in tetanic tension. In BC preparations, the methanolic fraction did not affect contractures to exogenous acetylcholine or potassium chloride. Incubation with atropine showed there was certain modulation by prejunctional nicotinic receptors, whereas treatment with nifedipine showed that the neurofacilitation required the entry of extracellular calcium. Tetrodotoxin did not prevent the facilitatory effect of 3,4-DAP or neostigmine, but antagonized the response to the methanolic fraction. These findings indicate that neuronal sodium channels have an important role in the facilitatory response to the methanolic fraction, with extracellular calcium entry via calcium channels modulating this neurofacilitation. Possible modulation of prejunctional cholinoceptors was not excluded, particularly in view of certain antagonism by the methanolic fraction at muscarinic receptors. Since facilitation by the methanolic fraction involved enhanced acetylcholine release, use of this fraction could be potentially beneficial in neuromuscular diseases and in the reversal of residual paralysis in the post-operative period or after local anaesthesia.

An isoflavone from Dipteryx alata Vogel is active against the in vitro neuromuscular paralysis of Bothrops jararacussu snake venom and bothropstoxin I, and prevents venom-induced myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 µg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 µg/mL) caused irreversible paralysis. Preincubation of TM (200 µg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.