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.