Molecular modulation of cholinergic and neuromuscular signaling following long-term acetaminophen administration

Acetaminophen is widely used as an analgesic and antipyretic; however, its long-term neurochemical actions remain incompletely characterized. Its bioactive metabolite, N-arachidonoylphenolamine (AM404), activates transient receptor potential vanilloid type 1 (TRPV1) and cannabinoid receptor type 1 (CB1), both of which are involved in the regulation of cholinergic signaling. This study investigated whether chronic acetaminophen exposure modulates cholinergic and neuromuscular-related molecular signaling associated with the TRPV1-CB1-acetylcholine (ACh) axis. Human neural stem cells and mouse myoblast cells were treated with AM404 (0-1.0 μg/mL), and Sprague-Dawley rats received oral acetaminophen (0, 10, 100, or 1000 mg/kg/day) for 4 weeks. In vitro, AM404 increased the expression of TRPV1, CB1, choline transporter (CHT), choline acetyltransferase (ChAT), and vesicular ACh transporter (VAChT) in F3 cells. Meanwhile, it upregulated CHRNA1, ryanodine receptor 1 (RyR1), and sarco/endoplasmic reticulum Ca²⁺-ATPase 1 (SERCA1) in C2C12 cells. In vivo, chronic acetaminophen administration elevated TRPV1, CB1, CHT, ChAT, and VAChT in the brain and sciatic nerve, increased cerebrospinal ACh concentrations, and increased expression of CHRNA1, RyR1, and SERCA1 in skeletal muscle. Hepatotoxicity was observed only at the highest dose (1000 mg/kg), whereas at lower doses molecular changes in cholinergic and neuromuscular-related markers were observed without overt liver injury. These observations provide new insight into the neuromodulatory properties of acetaminophen and warrant further investigation in disease-relevant models.