Effect of positively charged local anesthetics on a membrane-bound phosphatase in Acholeplasma laidlawii.

The plasma membrane p-nitrophenylphosphatase activity of Acholeplasma laidlawii was stimulated by the spin-labeled local anesthetic 2-[N-methyl-N-(2,2,6,6-tetramethylpiperidinooxyl)]ethyl p-hexyloxybenzoate, abbreviated as C6SL, and its methylated quaternary analog, C6SLMeI. The tertiary amine C6SL (at a concentration of 5 X 10(-5) M) was more potent at pH 6.5 than at pH 7.7. In contrast, the permanently-charged C6SLMeI was equally potent, independently of pH. These results suggest that cationic forms of the anesthetics are responsible for stimulating the enzyme. Electron spin resonance studies of C6SL- and C6SLMeI-labeled membranes showed that these anesthetics in their cationic forms interacted electrostatically with components of the Acholeplasma membrane. For C6SL, this interaction was pH dependent and correlated with the pH dependency of the anesthetic-induced enzyme stimulation in the Acholeplasma membranes. Further, studies using 5-doxylstearic acid labels and non-spin-labeled anesthetics at various pH values showed that the membrane-fluidizing effect of anesthetics was not correlated with anesthetic-induced pNPPase stimulation. Our observations are consistent with the hypothesis that electrostatic interactions between cationic local anesthetics and anionic membrane components may lead to functional changes mediated by membrane proteins.