Title:The Facilitatory Effect of Casearia sylvestris Sw. (guaçatonga) Fractions on the Contractile Activity of Mammalian and Avian Neuromuscular Apparatus
Volume: 16
Issue: 5
Author(s): Adriana C. Werner, Miriele C. Ferraz, Edson H. Yoshida, Natalia Tribuiani, Jean A.A. Gautuz, Monique N. Santana, Bruna A. Dezzotti, Vanessa G. de Miranda, Ameris L. Foramiglio, Sandro Rostelato-Ferreira, Renata V. da Silva Tavares, Stephen Hyslop and Yoko Oshima-Franco
Affiliation:
Keywords:
Casearia sylvestris, chick biventer cervicis, guaçatonga, mouse phrenic nerve-diaphragm preparation, neuromuscular
junction.
Abstract: 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.