In Situ Effects of Doxycycline on Neuromuscular Junction in Mice

Abstract

Background: The antibacterial mechanism of doxycycline is known, but on the nerve-muscle apparatus is yet unclear. Objective: To combine molecular targets of the neuromuscular machinery using the neuronal blocker effect doxycycline, a semisynthetic second-generation tetracycline derivative, on mice neuromuscular preparations, in situ. Methods: Doxycycline was assessed at the neurotransmission; presynaptic; synaptic cleft; and postsynaptic, including the muscle fiber, using the traditional myographic technique. Preliminarily, doxycycline showed an "all or nothing" effect, being "all" obtained with 4 µM and "nothing", with 1-3 µM. The rationale of this study was to apply known pharmacological tools against the blocker effect of 4 µM doxycycline such as F55-6 (Casearia sylvestris), CaCl2 (or Ca2+), atropine, neostigmine, polyethylene glycol (PEG 400), and d-Tubocurarine. The evaluation of cholinesterase enzyme activity, the diaphragm muscle histology, and protocols on the neuromuscular preparation submitted to indirect or direct stimuli were complementary. Results: Doxycycline does not affect cholinesterase activity nor cause damage to skeletal muscle diaphragm; acts on ryanodine receptor, sarcolemmal membrane, and on neuronal sodium channel with a postjunctional consequence due to the decreased availability of muscle nicotinic acetylcholine receptors. Conclusions: In conclusion, using the blocker effect we showed that doxycycline acts on multiple targets, among them, is antagonized by F55-6, a neuronal Na+-channel agonist and Ca2+, but not by neostigmine.

Multi targets of cannabidiol (CBD) on skeletal mammalian and avian neuromuscular preparations

“…For further assays, we selected the latter concentration due to the homogeneity of responses. The role of an excess of calcium (Tribuiani et al 2022) using 1.8 mM Ca 2+ (Sigma®) on CBD 0.3 mM was investigated.…”

“…The BC preparations were stabilized for at least 10 min before the addition of 40 μM acetylcholine (ACh) for 60 seconds (s) or 100 mM potassium chloride (KCl) for 60 s. Contractures to exogenous ACh or KCl were recorded in the absence of field stimulation before or after the end of the experiment as a test for evaluating pre-or post-synaptic action and, at the same time, the integrity of the sarcolemma. Atropine, a non-selective muscarinic antagonist (2 µg/mL, Sigma-Aldrich Co., St. Louis, Missouri); polyethylene glycol 400 (PEG 400, 20 µg/mL, Synth®, Diadema, SP, Brazil) which acts on the sarcolemmal membrane at the triad level (Oshima et al 2010), F55-6 (from Casearia sylvestris), an agonist of the neuronal sodium channel (Ferraz et al 2012, Tribuiani et al 2022, Werner et al 2015, and nifedipine (Wali 1986) (Sigma-Aldrich Co, St. Louis, Missouri), an L-type Ca 2+ channel blocker were used.…”