Garret Vanderkooi

Multiple sites of inhibition of mitochondrial electron transport by local anesthetics

Local anesthetics and alcohols were found to inhibit mitochondrial electron transport at several points along the chain. THe anesthetics employed were the tertiary amines procaine, tetracaine, dibucaine, and chlorpromazine, and the alcohols were n-butamol, n-pentanol, n-hexanol, and benzyl alcohol. Uncoupled sonic submitochondrial particles from beef heart and rat liver were studied. We report the following: (1) All of the anesthetics were found to inhibit each of the segments of the electron transport chain assayed; these included cytochrome c oxidase, durohydroquinone oxidase, succinate oxidase, NADH oxidase, succinate dehydrogenase, succinate-cytochrome c oxidoreductase, and NADH-cytochrome c oxidoreductase. (2) NADH oxidase and NADH-cytochrome c oxidoreductase required the lowest concentration of anesthetic for inhibition, and cytochrome c oxidase required the highest concentrations. (3) We conclude that there are several points along the chain at which inhibition occurs, the most sensitive being in the region of Complex I (NADH dehydrogenase). (4) Beef heart submitochondrial particles are less sensitive to inhibition than are rat liver particles. (5) Low concentrations of several of the anesthetics gave enhancement of electron transport activity, whereas higher concentrations of the same agents caused inhibition. (6) The concentrations of anesthetics (alcohol and tertiary amine) which gave 50% inhibition of NADH oxidase were lower than the reported concentrations required for blockage of frog sciatic nerve.

Further studies on F1-ATPase inhibition by local anesthetics

We have measured the inhibitory potencies of several local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and related compounds (chlorpromazine, procainamide and propranolol) on the ATPase activities of bovine heart submitochondrial particles and purified F1 extracted from these particles. All of these agents cause inhibition of ATPase in F1 as well as in submitochondrial particles. A linear relationship is found between the log of the octanol/water partition coefficients and the log of the concentrations required for 50% inhibition of F1. Sedimentation velocity ultracentrifugation and polyacrylamide gel electrophoresis showed that 1.0 mM tetracaine caused partial dissociation of the F1 complex. Complete reversibility of the enzyme inhibitory effects was demonstrated, however. This work shows that local anesthetics can affect protein structure and enzyme activity without the mediation of lipid.

Molecular origin of the internal dipole potential in lipid bilayers: calculation of the electrostatic potential

The finite difference linearized Poisson-Boltzmann equation was solved for a segment of bilayer for two lipids (phosphatidylcholine dihydrate and phosphatidylethanolamine-acetic acid) in order to obtain the transbilayer electrostatic potential. Atomic coordinates derived from the crystal structures of these lipids were used, and partial changes were assigned to all atoms in the polar parts of the molecules. These calculations confirmed that a dipole potential exists in the uncharged hydrophobic interior of a bilayer. The phosphocholine and phosphoethanolamine groups make negative contributions to the internal potential, and the glycerol acyl esters make positive contributions, but the sum of these terms is negative. The water of hydration in phosphatidylcholine, and the acetic acid which is present in the phosphatidylethanolamine crystal structure, make positive contributions to the internal potential. It is concluded that the water of hydration in fully hydrated lipid bilayers is mainly responsible for the experimentally inferred positive sign of the internal potential.

Electrochemical properties of spherical polyelectrolytes: I. Impermeable sphere model

Abstract The Poisson-Boltzmann equation was numerically solved to obtain the electrostatic potential in the vicinity of spherical polyelectrolyte particles at finite concentration and low ionic strength, using the cell model. The polyelectrolyte particles were treated as impermeable spheres having a uniform surface charge density. The electrostatic potential was studied as a function of surface charge density, concentration of added salt, and particle size. The osmotic coefficient, Δp K , and the local ion concentrations were computed from the electrostatic potential. The computational results were compared with experimental ionic activities and osmotic coefficients for micelles of ionic detergents. Excellent agreement was obtained between the experimental and computed osmotic coefficients for micelles of sodium dodecyl sulfate and potassium laurate, but not for the cationic detergents dodecylpyridinium chloride and dodecylammonium chloride. The reason for the disagreement is unknown. The present calculations at finite particle concentration are compared with the electrostatic potential tables of Loeb et al. (“The Electrical Double Layer around a Spherical Colloid Particle,” M.I.T. Press, Cambridge, Mass., 1961), which were computed for the special case of infinite dilution or high ionic strength in order to show the range of applicability of those tables. Detailed comparison requires the specification of all parameters; for the particular parameters used in this paper it was found that for particles having the radius of detergent micelles (∼18 A), the presence of 0.1 M salt was required to give good agreement (within 1.5%) between the surface potential computed by the present method and the infinite-dilution method, whereas only 10 −4 M salt was needed to give similarly good agreement for particles having the radius of phospholipid vesicles (∼300 A).

On the mechanism of action of anesthetics. Direct inhibition of mitochondrial F1-ATPase by n-butanol and tetracaine.

The concentrations of n-butanol and tetracaine required for 50% inhibition of the ATPase activity of F1 particles isolated from bovine heart mitochondria were 160 mM and 1.1 mM, respectively. The results are offered as evidence that the physiological effects of these anesthetics may be due to direct interaction with membrane proteins rather than with the lipids.

Inhibition of synaptosomal enzymes by local anesthetics

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na+ + K+)-ATPase (ouabain-sensitive), Mg2+-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPases, for example 7.9 and 10 mM tetracaine for Mg2+-ATPase and (Na+ + K+)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F1-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.

Computation of mixed phosphatidylcholine-cholesterol bilayer structures by energy minimization.

The energetically preferred structures of dimyristoylphosphatidylcholine (DMPC)-cholesterol bilayers were determined at a 1:1 mole ratio. Crystallographic symmetry operations were used to generate planar bilayers of cholesterol and DMPC. Energy minimization was carried out with respect to bond rotations, rigid body motions, and the two-dimensional lattice constants. The lowest energy structures had a hydrogen bond between the cholesterol hydroxyl and the carbonyl oxygen of the sn-2 acyl chain, but the largest contribution to the intermolecular energy was from the nonbonded interactions between the flat alpha surface of cholesterol and the acyl chains of DMPC. Two modes of packing in the bilayer were found; in structure A (the global minimum), unlike molecules are nearest neighbors, whereas in structure B (second lowest energy) like-like intermolecular interactions predominate. Crystallographic close packing of the molecules in the bilayer was achieved, as judged from the molecular areas and the bilayer thickness. These energy-minimized structures are consistent with the available experimental data on mixed bilayers of lecithin and cholesterol, and may be used as starting points for molecular dynamics or other calculations on bilayers.

DIBUCAINE FLUORESCENCE and LIFETIME IN AQUEOUS MEDIA AS A FUNCTION OF pH

Abstract— The absorption and fluorescence spectra and fluorescence lifetimes of the local anesthetic dibucaine (2‐butoxy‐N‐[2‐(diethylamino) ethyl]‐4‐quinoline carboxamide) are reported as a function of pH in aqueous media. The fluorescence emission maxima are at 400 nm in neutral solution, 397 nm in alkali, and 454 nm in acid. The fluorescence is strongest and has the longest lifetime in neutral solution; the quantum yields are 0.25, 0.033, and 0.032, and the lifetimes (determined by the phase shift method) are 3.29, 0.60, and 1.37 ns, for the neutral, alkaline, and acid solutions, respectively. The pKoi the tertiary amine group was determined by fluorescence intensity and lifetime measurements to be 8.95, and the pK of the aromatic nitrogen was found to be 1.80 ± 0.03 by absorption and fluorescence intensity measurements. Phase and modulation lifetimes differed in the manner expected for a two lifetime system in the pH titration range of the tertiary amine, but gave anomalous results in the titration range of the aromatic nitrogen.

Electrochemical properties of spherical polyelectrolytes: II. Hollow sphere model for membranous vesicles

The nonlinearized Poisson-Boltzmann equation has been numerically solved to obtain the electrostatic potential inside and outside of hollow spherical shells suspended in an aqueous medium. The geometrical characteristics of the shells were chosen to correspond to those of the small (100 to 600 A radius) vesicles typically formed by sonication of phospholipids or membranes. The boundary conditions were specified using the following assumptions: (i) The shells are permeable to ions, permitting an electrochemical equilibrium to exist between the inner and outer solutions, (ii) Both inner and outer surfaces of the shell bear ionizable groups, but only the total (in plus out) degree of dissociation is given, (iii) The cell model for finite concentrations of vesicles was employed. The additional assumption of electroneutrality within the vesicles was used in most of the reported calculations. It is shown that for vesicles having radii in the range studied here, the computed results obtained with and without the electroneutrality constraint are nearly the same. The electrostatic potential was studied as a function of salt concentration, vesicle concentration, shell thickness, radius of shell, and surface area per ionizable group. The ionic distribution, activity coefficients, Δp K , and osmotic coefficient were obtained from the electrostatic potential. The degree of dissociation under all circumstances was found to be less on the inside than the outside. The average potential of the inside solution is larger in magnitude than that outside the shell at low salt and polyelectrolyte concentrations. At high salt concentration the potentials are similar inside and outside, whereas at high polyelectrolyte concentration, but low salt, the magnitude of the potential is larger outside than inside.

Local anesthetics: a new class of partial inhibitors of mitochondrial ATPase.

The following characteristics are reported for mitochondrial ATPase prepared by the chloroform extraction method: (1) The pH optimum for enzyme activity is at 8.0. (2) The neutral anesthetic benzocaine inhibits the enzyme at all pH values. (3) Reciprocal plots of 1/v versus 1/[ATP] show that inhibition by lidocaine, tetracaine, dibucaine, and chlorpromazine is noncompetitive; slope and intercept replots are hyperbolic, showing that the inhibition is partial rather than complete.