Ontogenic expression of acetylcholinesterase activity in trachealis of young swine.
Previous investigations have demonstrated that cholinergic contraction of porcine tracheal smooth muscle (TSM) decreases between the second and tenth weeks of life. In this investigation, we hypothesized that the greater contractile response to acetylcholine (ACh) in TSM of 2-wk-old swine (2ws) vs. 10-wk-old swine (10ws) was the result of a relative decrease in activity of acetylcholinesterase (AChase). To examine this hypothesis, we assessed AChase activity directly in homogenates of TSM from eight 2ws and seven 10ws using a newly adapted method that measures the rate of cleavage of acetylthiocholine; enzyme activity was expressed as absorbance units per minute per milligram protein. The AChase from tissues of both age groups saturated at approximately 3 mM substrate. However, maximal AChase activity (Vmax) was significantly greater in 10ws than 2ws. Eadie-Hofstee analysis of enzyme kinetics revealed similar Michaelis-Menten constants for 2ws and 10ws. The concentration of physostigmine (PS), an inhibitor of cholinesterase, that elicited half-maximal inhibition of AChase activity also was similar for 2ws and 10ws. In separate studies, contraction of TSM strips was assessed in vitro at optimal resting length and expressed as a function of maximal force generation to potassium chloride. Strips of TSM from 2ws contracted with greater force than those of 10ws. After pretreatment with 10(-8) M PS, contractile forces were similar in 2ws and 10ws. We conclude that AChase activity measured directly in muscle homogenates is significantly reduced in TSM of 2ws vs. 10ws and that this may result in augmented contraction to ACh under conditions of zero-order kinetics.
Murphy, TM; Mitchell, RW; Phillips, IJ; Leff, AR
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