Overton's Rule Research Articles

Ecotoxicology of narcosis: Stereoselectivity and potential target sites

The stereoselectivity of certain anesthetics is currently thought to be inconsistent with lipid theories of narcosis. The EC 50-values of etomidate enantiomers for tadpole narcosis are now examined as a function of octanol/water partition coefficients, and enhancement factors for predicted over experimental EC 50 values are determined from a calibration curve for non-selective narcosis. The unfavored S-(−)-enantiomers of etomidate and two analogues surprisingly still fulfill the Meyer–Overton rule. The R(+)-enantiomers of etomidate and four structural analogues are up to 34-fold more active than expected. The non-chiral anesthetic, propofol, is 8-fold more active than expected. It is concluded that there may be two pathways to tadpole narcosis: enhanced narcosis involving specific receptor binding sites and non-selective narcosis corresponding to the Meyer–Overton rule and operating on the lipid/protein interface.

Theory of passive permeability through lipid bilayers.

Recently measured water permeability through bilayers of different lipids is most strongly correlated with the area per lipid A rather than with other structural quantities such as the thickness. This paper presents a simple three-layer theory that incorporates the area dependence in a physically realistic way and also includes the thickness as a secondary modulating parameter. The theory also includes the well-known strong correlation of permeability upon the partition coefficients of general solutes in hydrocarbon environments (Overton's rule). Two mathematical treatments of the theory are given; one model uses discrete chemical kinetics and one model uses the Nernst-Planck continuum equation. The theory is fit to the recent experiments on water permeability in the accompanying paper.

How fatty acids of different chain length enter and leave cells by free diffusion

Opposing views exist as to how unesterified fatty acids (FA) enter and leave cells. It is commonly believed that for short- and medium-chain FA free diffusion suffices whereas it is questioned whether proteins are required to facilitate transport of long-chain fatty acid (LCFA). Furthermore, it is unclear whether these proteins facilitate binding to the plasma membrane, trans-membrane movement, dissociation into the cytosol and/or transport in the cytosol. In this mini-review we approach the controversy from a different point of view by focusing on the membrane permeability constant ( P) of FA with different chain length. We compare experimentally derived values of the P of short and medium-chain FA with values of apparent permeability coefficients for LCFA calculated from their dissociation rate constant ( k off), flip-flop rate constant ( k flip) and partition coefficient ( K p) in phospholipid bilayers. It was found that Overton's rule is valid as long as k flip≪ k off. With increasing chain length, the permeability increases according to increasing K p and reaches a maximum for LCFA with chain length of 18 carbons or longer. For fast flip-flop (e.g. k flip=15 s −1), the apparent permeability constant for palmitic acid is very high ( P app=1.61 cm/s). Even for a slow flip-flop rate constant (e.g. k flip=0.3 s −1), the permeability constant of LCFA is still several orders of magnitude larger than the P of water and other small non-electrolytes. Since polyunsaturated FA have basically the same physico-chemical properties as LCFA, they have similar membrane permeabilities. The implications for theories involving proteins to facilitate uptake of FA are discussed.

One hundred years of membrane permeability: does Overton still rule?

The Overton Rule states that entry of any molecule into a cell is governed by its lipid solubility. Overton's studies led to the hypothesis that cell membranes are composed of lipid domains, which mediate transport of lipophilic molecules, and protein 'pores', which transport hydrophilic molecules. Recent studies, however, have shown that hydrophobic molecules are also transported by families of transporter proteins.

The effects of temperature and pressure on the thermodynamic activity of anesthetics

Anesthetic potency is often expressed by volume percent or partial pressure in the gas phase, concentrations in the aqueous phase, etc. However, these values do not represent the anesthetic activity at the action sites. Because the activity at the action sites is difficult to obtain, Ferguson (Ferguson, J., 1939. Proc. R. Soc. Lond. B 127 387–404) defined the thermodynamic activity, which is the ratio between the anesthetizing partial pressure and the vapor pressure of the pure anesthetic at the same temperature. This paper discusses the effects of temperature and pressure on the thermodynamic activity of anesthetics. It also discusses the limitations of the Meyer–Overton rule.

A hypothesis about the endogenous analogue of general anesthesia.

The advent of general anesthesia remains one of the most important advances in the history of medicine. Although the first popular demonstration of general anesthesia occurred more than 150 years ago at the now-famous ether dome of the Massachusetts General Hospital, the mechanism by which it occurs is still debated (1). Surprisingly, among all of the questions asked about general anesthesia, the important issue of if there is an endogenous analogue of the process is rarely discussed. This situation is in distinction to the usual course of events when an exogenous natural product is found to have biological activity in that much activity is devoted to a search for the endogenous counterpart. Think, for example, about the opiates and the endorphins. As a formal proposition the question is if the brain or body makes compounds that have the properties and mode of action of general anesthetics. The issue reduces to what, if any, natural process are the general anesthetics mimicking. This question surfaces now because of the recent discovery of a sleep-inducing lipid, oleamide, in the cerebrospinal fluid (CSF) of sleep-deprived cats (2, 3). Oleamide since has been shown to affect diverse membrane proteins (4), has a structure expected to perturb the fluidity of membrane lipids, and is accompanied by a membrane-bound brain enzyme that rapidly inactivates the compound (3, 5). It is therefore possible that the way oleamide operates is unusual in that it perturbs lipid matrices either free or complexed to proteins and protein assemblies. This concept is worth considering because it would represent a new form of regulation distinct from the usual receptor-ligand interaction. The main chemical facts about general anesthetics concern their diverse nature, hydrophobicity, and general lack of stereospecificity. The oldest correlate is called the Meyer–Overton rule, which …

Permeability properties of rat renal lysosomes.

Although lysosomes maintain large pH gradients and may be subjected to significant osmotic gradients in vivo, little is known about their passive permeability properties. In recent studies, vacuolar H(+)-adenosine-triphosphatases (ATPases), such as those found in lysosomes, have been suggested to act as water channels. In addition, the erythrocyte and proximal tubule water channel CHIP28 is present on the plasma membrane of proximal tubule cells and may undergo endocytosis so that it is incorporated in lysosomes. We therefore examined water, proton, and small nonelectrolyte permeabilities in freshly purified lysosomes from rat renal proximal tubule. Lysosomes were purified by differential and Percoll gradient centrifugation. The preparation contained only lysosomes when examined by electron microscopy. Moreover, analysis by flow cytometry showed virtually all particles to be positive for acid phosphatase and cathepsin B activities. Permeabilities were measured on a stopped-flow fluorimeter by monitoring the self-quenching or pH-sensitive quenching of entrapped fluorescein derivatives. Osmotic water permeability (Pf) averaged 0.011 +/- 0.003 cm/s (n = 6), a value similar to that of biological membranes containing water channels. However, Pf was insensitive to the organic mercurial reagent p-chloromercuribenzene-sulfonate and to HgCl2 and exhibited an activation energy of 10.8 +/- 0.8 kcal/mol. These results indicate that water flux in lysosomes occurred via the lipid bilayer, and not via water channels. Addition of ATP led to lysosomal acidification (proton flux = 4.6 +/- 0.8 x 10(-11) mmol H+.s-1.cm-2), which was completely inhibited by 0.1 microM bafilomycin. Pf was insensitive to this agent as was the passive proton permeability (0.36 +/- 0.18 cm/s, n = 4). Permeabilities to small nonelectrolytes varied in proportion to the oil-water partition coefficient, confirming the applicability of Overton's rule to lysosomes. We conclude that proximal tubular lysosomes exhibit high Pf, which occurs via the lipid bilayer and not via vacuolar H(+)-ATPase.

Apical membrane of the gastric parietal cell: water, proton, and nonelectrolyte permeabilities.

Gastric parietal cell apical membranes must protect the cell from the extremely low pH and wide variations in osmolality of the gastric juice. To characterize the permeability properties of gastric apical membranes, we have measured passive permeabilities to water, protons, NH3, and small nonelectrolytes of membrane vesicles derived from parietal cells of fasted animals and fed animals. Both preparations are known to be highly enriched in H+/K(+)-ATPase, the enzyme responsible for acidifying the gastric contents. The preparations behaved as single populations, and their permeability properties were similar in all respects, permitting pooling of the results. This similarity suggests that insertion of tubulovesicles into the apical membrane does not change the behavior of the lipid bilayer. Osmotic water permeability (Pf) averaged (mean +/- SD) (2.8 +/- 0.3) x 10(-4) cm/s, a value 10-fold lower than that obtained in lecithin large unilamellar vesicles (LUV) and similar to that obtained in other water-tight epithelia. Similarly, ammonia permeability (PNH3) was low [(4.4 +/- 2.3) x 10(-3) cm/s] and 10 times below that of lecithin LUV. By contrast, proton permeability (PH+) was surprisingly high (0.030 +/- 0.011 cm/s) and similar to that of lecithin LUV. These results suggest that the pathway for proton permeation differs from that of water and NH3. Nonelectrolyte permeabilities were strikingly similar to those obtained in another water-tight epithelium, the toad urinary bladder. Moreover, these permeabilities followed Overton's rule in that permeability varied in accordance with the oil-water partition coefficient. We conclude that the gastric apical membrane, like that of several renal epithelia, is relatively water-tight and exhibits low permeabilities to small nonelectrolytes. These properties are likely to be essential to the ability of this membrane to perform its barrier function.

Water and nonelectrolyte permeabilities of apical membranes of toad urinary bladder granular cells.

Certain types of epithelial cells such as those lining the toad urinary bladder have been classified as "tight" because their apical membranes exhibit low permeabilities to water, ions, and small nonelectrolytes. However, the permeability properties and structural features of these specialized apical membranes remain unclear because these membranes have never been purified. To isolate toad bladder granular cell apical membranes, we derivatized the bladder apical surface with the membrane-impermeant bifunctional reagent N-hydroxysulfosuccinimydyl-S,S-biotin (NHS-SS-biotin). After cell disruption, these derivatized apical membranes were purified using streptavidin-coated magnetic beads in a magnetic field. With the use of lactoperoxidase-mediated radioiodination as a marker for apical membrane, this preparative procedure purified apical membrane 48- or 72-fold as compared with homogenate. Thin section electron microscopy revealed unilamellar vesicles with some nonvesiculated membranes, while fragments of organelles such as mitochondria were absent. Water and nonelectrolyte permeabilities of purified apical membrane vesicles were similar to those obtained in intact bladders in the absence of antidiuretic hormone stimulation. The results demonstrate that isolated apical vesicles do not contain water channels and confirm the applicability of Overton's rule to the apical membrane of the toad urinary bladder. The technique has general applicability to isolation of other plasma membranes, and the apical membranes obtained are suitable for structural analysis.

Amino acid permeability of liposomal membranes measured by light scattering method: a simulation with a method of cumulants

Four types of liposomes were formed from egg lecithin (PC), PC-cholesterol (2:1) (PC-Chol), PC-phosphatidic acid (9:1) (PC-PA) and PC-dicetyl phosphate (9:1) (PC-DCP). The purity of PC used was 88% and it included 9% lysolecithin. Observations of the light scatter 90° to incident indicates that the liposomes exposed to a hypertonic medium of amino acid undergo rapid shrinkage due to water loss and that a slower re-swell phase, dependent on amino acid entry into the intraliposomal spaces, follows. Taking into account the polydispersity of liposome size and its property, the volume dependent change was simulated with a method of cumulants. In PC liposomes, the order of rate constant for influx was Ala, Gly < Ser < His, Pro < Val < Leu < Phe and Overton's rule was held for amino acids. Their relative magnitudes were parallel with the hydrophobic scale of amino acids given by Nozaki and Tanford (1971). A 10-fold increase in the rate constant accompanied a —1430 cal/mol change in the additional free-energy of transfer from water to organic solvent. The PC-Chol, PC-PA and PC-DCP membranes had lower rate constants for amino acids in comparison with that of PC liposomes. Water permeability of PC-Chol membrane was lower than that of PC. Inclusion of cholesterol into lecithin showed its condensing and fluidizing effects. The addition of phosphatidic acid which has a higher transition temperature in comparison with that of lecithin increased water permeability. This was explained from a phase separation effect which occurs when two lipids with different transition temperatures are mixed.

The nonelectrolyte permeability of planar lipid bilayer membranes.

The permeability of lecithin bilayer membranes to nonelectrolytes is in reasonable agreement with Overton's rule. The is, Pd alpha DKhc, where/Pd is the permeability coefficient of a solute through the bilayer, Khc is its hydrocarbon:water partition coefficient, and D is its diffusion coefficient in bulk hydrocarbon. The partition coefficients are by far the major determinants of the relative magnitudes of the permeability coefficients; the diffusion coefficients make only a minor contribution. We note that the recent emphasis on theoretically calculated intramembranous diffusion coefficients (Dm'S) has diverted attention from the experimentally measurable and physiologically relevant permeability coefficients (Pd'S) and has obscured the simplicity and usefulness of Overton's rule.

The membrane binding of morphine, diphenylhydantoin, and tetrahydrocannabinol.

The adsorption of morphine to guinea pig brain synaptosome membranes and to human erythrocyte membranes was found to be passive, and unaffected by time, temperature, ouabain, dinitrophenol, saponin, or ATP. The membrane/buffer partition coefficient for morphine was 35 (at low ionic strength) and 1.2 (at high ionic strength). The synaptosome membrane/buffer partition coefficient for diphenylhydantoin was around 60 (at pH 8), while that for Δ9-tetrahydrocannabinol (THC) was around 380 (in the concentration range of around 10−5 M). The partition coefficient for the latter drug dropped by a factor of two or three with increasing drug concentrations for both erythrocyte ghosts and synaptosomes; there may be two types of binding sites for THC. The minimum blocking concentrations (frog sciatic nerve) were 1.1 × 10−2 M for morphine, and 8.3 × 10−4 M for diphenylhydantoin. The anesthetizing membrane concentrations of these two drugs are close to the value predicted by the Meyer–Overton rule for local anesthesia (30 mmol/kg dry membrane).