INHIBITION OF RAT CHOLINESTERASE ISOENZYMES IN VITRO AND IN VIVO BY THE POTATO ALKALOID, ?-CHACONINE2

Abstract

The inhibition of purified bovine erythrocyte acetylcholinesterase (AchE) and horse serum cholinesterase (ChE) by α-chaconine was found to be a mixed-type with inhibition constants (Ki) for both enzymes of 8.3 × 10-6 M and 4.0 × 10-4 M, respectively. Kinetic constants for AchE obtained for the hydrolysis of acetylthiocholine iodide averaged 7.2 × 10-5 moles/liter/min (Vmax) and 6.2 × 10-5 M (Km) whereas for ChE these were 3.8 × 10-4 moles/liter/min (Vmax) and 1.3 × 10-4 M (Km) for the hydrolysis of butyrylthiocholine iodide. Subcellular acetylcholinesterase activity in nuclear-free rat brain homogenate showed the highest activity to be equally distributed between the mitochondrial and microsomal fractions, with only little activity in the nuclear fraction. Inhibition of the rat brain subcellular enzyme activity in vitro by 0.016 M α-chaconine was 43% for whole homogenate, 55% for nuclear fraction, 35% for mitochondria, and 33% for microsomes. In vivo administration of α-chaconine, (10, 30 and 60 mg/kg) to adult male rats caused a reduction of brain AchE activity to 79, 55 and 18% of the control activity for the respective doses. Heart and plasma AchE activity of treated animals did not show a dose-related inhibition similar to that of brain. Inhibition of heart AchE was 61%, while that of plasma was 51% for rats administered to 10 mg/kg and no further inhibition for rats given 30 mg/kg α-chaconine was noted. Isoenzymes of rat brain cholinesterases, separated by acrylamide gel electrophoresis, were inhibited by 30 and 60 mg/kg α-chaconine administered intraperitoneally. Electrophoretic separation of plasma from the treated animals resulted in five anodally migrating zones that hydrolyzed acetylthiocholine and α-naphthylacetate. Inhibition of the activity of isoenzyme bands 1 and 2 was 40 and 77% respectively for animals given 10 mg/kg α-chaconine, while rats given 30 mg/kg showed inhibition of 100 and 75%, respectively. Isoenzyme bands 3 and 4 were completely inhibited in the plasma of alkaloid treated animals. In vitro inhibition of rat plasma, erythrocyte and brain esterase isoenzymes was estimated by incubating gels with 10-4 M α-chaconine after electrophoretic separations. The slower-moving isoenzyme bands were inhibited completely, while the faster migrating isoenzyme bands in plasma and erythrocytes were least affected by the concentration of α-chaconine used.