Local anesthetics and pressure: a comparison of dibucaine binding to lipid monolayers and bilayers

Abstract

The binding of the local anesthetic dibucaine to monolayers composed of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine was studied with a Langmuir trough at pH 5.5 (22° C, 0.1 M NaCl). At this pH value only the charged form of the local anesthetic exists in solution. Charged dibucaine was found to be surface active and to penetrate into the lipid monolayer, with the hydrophobic part of the molecule being accommodated between the fatty acyl chains of the lipid. The dibucaine intercalation could be quantitated by measuring the expansion of the film area, ΔA, at constant surface pressure, π. At a given surface pressure, ΔA increased with increasing dibucaine in the buffer phase. On the other hand, keeping the dibucaine concentration constant, the area increase, ΔA, was strongly dependent on the surface pressure. The area increase, ΔA, was large at low surface pressure and decreased with increasing surface pressure. A plot of the relative change in surface area, ΔA/3, versus the surface pressure yielded straight lines in the pressure range of 25–36 mN/m for five different concentrations. The ΔA/ A vs. π isotherms intersected at π = 39.5 − 1 mN/m with ΔA = 0, indicating that charged dibucaine apparently can no longer penetrate into the monolayer film. By making judicial assumptions about the area requirement of dibucaine the monolayer expansion curves could be transformed into true binding isotherms. Dibucaine binding isotherms were constructed for different monolayer pressures and were compared to a bilayer binding isotherm measured under similar conditions with ultraviolet spectroscopy. The best agreement between monolayer and bilayer binding data was obtained for a monolayer held at a pressure of 30.7 to 32.5 mN/m, which can thus be considered as the bilayer-monolayer equivalence pressure. It is further suggested from this analogy that the binding of dibucaine does not change the internal pressure in the bilayer phase, at least not in the concentration range of physiological interest (0–2 mM dibucaine) but induces a lateral expansion. At higher molar ratios of cationic dibucaine to lipid, x b, in the monolayer ( x b > 0.20) the area increase is larger than would be expected from the molecular dimensions of dibucaine. This is probably due to change repulsion effects, which at still higher molar ratios ( x b > 0.6) lead to a micellisation. The pressure dependence of the intercalation of cationic dibucaine into lipid membranes may also be of relevance for the phenomenon of pressure reversal in anesthesia.