OBJECTIVE: To investigate the role of reactive oxygen species (ROS) on cholinergic receptor function.
MATERIALS AND METHODS : Rectus abdominis and isolated heart preparations of frog (
Rana tigirina
) were used to assess nicotinic and muscarinic receptor activity, respectively. Thirty percent hydrogen peroxide (H
2O
2) solution and Fenton mixture (Fm, 13.9 mg, 50 µM of FeSO4
, 75 mg of sodium EDTA and 50 µL of 30% H
2O
2 were added to 10 ml of 0.1 M K
2HPO
4) were used to generate 1 mM H
2O2
and hydroxyl free radicals. The responses were recorded with acetylcholine at different phases of exposure of tissues to ROS. Normal frog Ringer was used as a physiological solution. Responses of acetylcholine were also recorded in the presence of ROS before and after exposure of the tissue to an antioxidant (ascorbic acid).
RESULTS : Free-radical-mediated receptor damage was dose (1-100 mM H
2O
2) and time (10-30 min) dependent when responses were taken with 30 µg and 30 ng of ACh for nicotinic and muscarinic receptors, respectively. There was no effect of ROS on prior exposure of tissue to ascorbic acid (antioxidant) at a concentration of 300 µg/ml. The antioxidant has not shown any beneficial effect on sulfhydryl groups of G-protein-coupled muscarinic receptors, which are more susceptible and sensitive to ROS than ion-channel nicotinic receptors where there is 96% protection with the antioxidant. Reactive oxygen species has shown different effects on receptor function.
CONCLUSION: Free radicals continuously cause considerable damage to the receptors. G-protein-coupled muscarinic receptors are more susceptible than ion-channel-linked nicotinic receptors. Antioxidants are shown to play a major role in protecting free-radical-mediated receptor damage.
