The effect of n-alkanols on the stationary current voltage behavior and action potential of myelinated nerve

Stationary current voltage characteristics and the action potential of single myelinated nerve fibres were measured to examine the effect of $\text{n-}\text{alkanols}$ (methanol to octanol) on the electrophysiological function of the axon membrane. K⁺-depolarized membranes show alkanol-dependent shifts of $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$, the membrane transition voltage, whereas in veratridine-depolarized membranes such $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}\text{-}\text{shifts}$ are not observed. In the latter case, $\text{n-}\text{alkanols}$ reduce both the stationary Na⁺ current and the conductivity step between the high- and low-ohmic conductivity state of the membrane. Action potential amplitude, however, is less affected by the alkanols as is the stationary Na⁺ current. The results are compared with the alkanol-dependent changes of the thermotropic phase transition in phospholipid bilayers.     

Initial membrane reaction in peptidoglycan synthesis. Perturbation of lipid-phospho-N-acetylmuramyl-pentapeptide translocase interactions by n-Butanol

$\text{Phospho}\text{-N-}\text{acetylmuramyl-pentapeptide translocase}$, the initial membrane enzyme in the biosynthesis of peptidoglycan, requires a lipid microenvironment for function. $\text{n-}\text{Butanol}$ was reversibly intercalated into membranes to perturb the hydrophobic interactions in this microenvironment in order to define further the role of lipid. In the concentration range for maximal stimulation of enzymic activity (0.12–0.18 M), $\text{n-}\text{butanol}$ causes a 40% decrease in the fluorescence emission of the dansylated product, undecaprenyl $\text{diphosphate}\text{-(N}{}^{\mathrm{?}}\text{-}\text{dansyl)pentapeptide}$. Since no change in emission maximum occurs below 22°C in the presence of 0.12 M $\text{n-}\text{butanol}$, it is concluded that intercalation of this alkanol causes an increase in fluidity. Above 22°C this concentration of $\text{n-}\text{butanol}$ causes both a decrease in the fluorescence emission and a red shift in the emission maximum. It is concluded that a polarity change as well as fluidity change occurs above 22°C. $\text{n-}\text{Butanol}$ also causes a significant change in the phase transition experienced by the dansylated lipid product. Thus, it is possible with $\text{n-}\text{alkanols}$, e.g. $\text{n-}\text{butanol}$, to perturb lipid-translocase interactions resulting in an increase in fluidity in the microenvironment of the enzyme. This change in fluidity correlates with a stimulation of enzymic activity.     

Block of sodium conductance by n-octanol in crayfish giant axons

The block of the Na⁺ current by $\text{n-}\text{octanol}$ was studied in crayfish giant axons under axial wire voltage-clamp conditions. Standard kinetic analysis of the Na⁺ currents was undertaken to test the hypothesis that the $\text{n-}\text{octanol-induced}$ block of the Na⁺ current could be accounted for on the basis of changes in the voltage dependence of the kinetic parameters. Alterations in the membrane dipolar potential arising from rearrangement of membrane lipids would be the anticipated source of changes in the voltage dependence. Although some changes in voltage dependence did evolve with the block by $\text{n-}\text{octanol}$, the changes were not of sufficient magnitude to account for the block. In conclusion, although higher concentrations of $\text{n-}\text{octanol}$ produced shifts along the voltage axis of the kinetic parameters, direct blocking action of $\text{n-}\text{octanol}$ on the channel appears to be the most important mechanism of the block.     

Permeability changes of erythrocytes and liposomes by 5-(n-alk(en)yl) resorcinols from rye

$\text{5-(n-}\text{Alk(en)yl}\text{)}$ resorcinols can induce potassium release from liposomes and erythrocytes. The results suggest that $\text{5-(n-}\text{pentyl)resorcinol}$ can induce a specific permeability to protons as well as to potassium and other small molecules. The highest permeability changes were found in the presence of $\text{5-(n-}\text{pentadecyl)resorcinol}$ and alkenyl resorcinols. Orcin and resorcin were without effect. The size of permeant as investigated by turbidity measurements indicated that Ca²⁺ and Mg²⁺ cannot pass through the alkyl resorcinol-modified membrane but can pass through the alkenyl resorcinol-modified membrane. It was observed that alkenyl resorcinol at a concentration of 15 μM induced not only potassium release but also lysis of erythrocytes.     

A dynamic x-ray diffraction study of anaesthesia action. Changes in myelin structure and electrical activity recorded simultaneously from frog sciatic nerves treated with n-alkanes

Changes induced in the structure and electrical activity of myelin were recorded simultaneously from frog sciatic nerves treated with $\text{n-}\text{alkanes}$. The results suggest that the effect of $\text{n-}\text{alkanes}$ seems to be two-fold: (a) there is an initial reversible phase, in which a significant modification of the X-ray diffraction patterns, concomitant with the continuous fall of the action potential, is observed; (b) there is a final phase which is irreversible. This occurs some time after the complete abolition of the electrical activity. At this stage, further changes of the X-ray diffraction patterns are detected, the most significant of them being in the $\text{n-}\text{pentane-treated}$ myelin, and consist of an increase in the membrane bilayer thickness.     

Phospholipid phase transitions. Effects of n-alcohols, n-monocarboxylic acids, phenylalkyl alcohols and quatenary ammonium compounds

The interactions of a series of alcohols, acids and quaternary ammonium salts with a phosphatidylcholine-water model biomembrane (dipalmitoyl phosphatidylcholine) system have been studied using differential scanning calorimetry. In particular the effects of these molecules upon the lipid endothermic phase transitions were investigated over a range of concentrations. A variety of effects was observed. (a) Those molecules which shift or broaden the main lipid transition can also remove the pretransition endotherm. (b) $\text{n-}\text{Alcohols}$ and $\text{n-}\text{monocarboxylic}$ acids containing the same number of carbon atoms have very similar effects at molar concentrations up to 40%. Those molecules containing 12 or more carbon atoms raise the main lipid phase transition whilst those molecules containing 10 or less carbon atoms lower this transition temperature. (c) The phase diagram of stearoyl alcohol in the phosphatidylcholine-water system shows the formation of lipid-alcohol complexes. (d) Alkyl trimethyl ammonium bromides showed behaviour which differs considerably from $\text{n-}\text{alcohols}$ and $\text{n-}\text{carboxylic}$ acids of the same chain length. (e) Other alkyltrialkyl and tetraalkylammonium bromides show that a variety of effects on the lipid phase transition can be obtained. (f) With the homologous series of phenylalkyl alcohols from benzyl alcohol to 4-phenyl butanol increasing the number of methylenes between the terminal OH and the benzene ring leads to greater interaction between solute and bilayer.The range of different effects obtained with the compounds studied offers a means for introducing various degrees and types of perturbation into membrane systems.     

Comparison of the promoting activity of pristane and n-alkanes in skin carcinogenesis with their physical effects on micellar models of biological membranes

In 1976 (Horton, A.W., Butts, C.K. and Schuff, A.R. (1976) Colloid Interface Sci. 5, 159–168) we assayed pristance (2,6,10,14-tetramethylpentadecane) in a model interfacial system that has given excellent correlation with cocarcinogenic activity among $\text{n-}\text{alkanes}$, as tested in cycloalkane diluents. It was predicted that this branched-chain derivative of the diterpenes would have higher activity than $\text{n-}\text{C}{}_{18}\text{H}{}_{38}$, one of the most cocarcinogenic of the $\text{n-}\text{alkanes}$ in such diluents. Pristane was compared with $\text{n-}\text{C}{}_{18}\text{H}{}_{38}$ and two other $\text{n-}\text{alkanes}$ for their promoting activities in cyclohexane for C3H male mice after a single application of 7,12-dimethylbenz$\text{[a]}\text{anthracene}$. The branched-chain alkane proved to be more active. 20% $\text{n-}\text{tetracosane}$ in cyclohexane was inactive, which correlated with its effects in this diluent in the physical assay system. The promoting activity of 75% $\text{n-}\text{octane}$ in cyclohexane, predicted by the physical assay, was confirmed by tests on mice. The combined by-products of the synthesis of tetracosane, including C₁₂ alkanes and alkenes, C₁₉ and C₂₀ alkylbenzenes, and C₂₄ alkenes, proved to be a very active promoter. However, a mixture in cyclohexane of purified tetracosane with this composite of potential impurities was inactive. From the alkanes behavior in physical systems involving vatious membrane phospholipids and steroids, it is hypothesized that the primary step in their biological activity is a chain-chain interaction with membrane lipids that alters the properties of liquid-crystalline phases at aqueous interfaces. Resulting changes in the microfluidity of the lipid phase and the lateral mobility of critical hormone receptors and enzyme systems, such as the nucleotidyl cyclases, would perturb control systems that maintain the normal behavior of the initiated cell. Thus, its progression to a proliferating neoplasm may be favored.     

Electrophoretic measurements about the relation between transition voltage and ζ-potential of biological membranes

The effects of inorganic cations, $\text{n-}\text{hexanol}$, saccharose and ²H₂O on the electrophoretic mobility and ζ-potential of membrane vesicles from nerve myelin were measured and the results compared with the corresponding effects of the same reagents on the transition voltage, $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$, of the nerve axon membrane. Different cation concentrations and ²H₂O affect both potentials, the ζ-potential and $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$, in a parallel way. Saccharose and $\text{n-}\text{hexanol}$, however, shift $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$ but leave the electrophoretic mobility of the myelin vesicles unchanged. These results suggest that $\text{V}{}_{\mathrm{<mtext>Tr</mtext>}}$ shifts are not necessarily linked to changes in the membrane surface charge density but may also be caused by an interaction between the reagent and non-polar groups of the membrane interior.     

Effect of n-alkanes on membranes in lipid-water systems

The changes in leaflet thickness and surface area per lipid molecule as a function of added amount of $\text{n-}\text{alkane}$ solvents have been studied in a number of lipid water systems by low angle X-ray diffraction techniques. The most probable site of accumulation of the $\text{n-}\text{alkane}$ is identified as the middle of the leaflet as opposed to intercalation with lipid hydrocarbon chains. Adsorption of $\text{n-}\text{alkane}$ depends on alkyl chain length and the organisation of the lipid hydrocarbon chains in the lipid water phases. Attention is drawn to the possible relationship between these results, the effect of $\text{n-}\text{alkanes}$ on isolated lipid bilayers and their effects as anaesthetics.     

Release and purification of micrococcus lysodeikticus ATPase from membrane extracted with n-butanol

The ATPase associated with the membranes of Micrococcus ysodeikticus has been released into the aqueous phase (i.e. solubilized) by extracting the membranes with $\text{n-}\text{butanol}$ in a two-phase system modified from the procedure of Maddy, A.H. (164) Biochim. Biophys. Acta 88, 448–449. A procedure for the release and purification of the ATPase from the membranes extracted with $\text{n-}\text{butanol}$ is described as an alternate method to that previously used for the shock-wash ATPase. Upon extracting the membrane suspensions with $\text{n-}\text{butanol}$ the soluble ATPase released into the buffer phase no longer exhibits stimulation by trypsin in contrast to the shock-wash type of ATPase. As shown by Salton, M. R. J. and Schor, M. T. (1972) Biochem. Biophys. Res. Commun. 49, 350–357, the shock-wash ATPase possesses associated protein(s) as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis whereas these are absent from the purified ATPase released by the $\text{n-}\text{butanol}$ method. The specific activities of the purified ATPase released by the two methods were generally similar, the $\text{n-}\text{butanol}$ type being consistently somewhat higher.     

The effect of anaesthetic-like molecules on the phase transition in smectic mesophases of dipalmitoyllecithin I. The normal alcohol up to C = 9 and three inhalation anaesthetics

The effect of the normal alcohols (up to $\text{C = 9}$) and three clinically used anaesthetics, on the crystalline-liquid crystalline phase transition in $\text{1,2-}\text{dihexadecyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphorylcholine}$ have been studied. A one-degree depression was produced by a 4.4% concentration in the membrane of $\text{n-}\text{octanol}$ and $\text{n-}\text{nonanol}$ agreeing well with the value calculated from the temperature and enthalpy of the transition. It is also shown that the relationship between the partition coefficient $\text{P}$ and the water solubility $\text{S (P · S = 2)}$, holds for the solutes investigated here. The experimental method described offers a simple way of assessing the anaesthetic potency of a wide range of compounds.     

The effects of n-alcohols on sarcoplasmic reticulum vesicles

Short, mild treatments of sarcoplasmic reticulum vesicles with aqueous $\text{n-}\text{alcohols}$ from methanol to $\text{n-}\text{heptanol}$ caused an inhibition of calcium uptake and an enhancement of ATPase activity. The $\text{n-}\text{alcohol}$ treatments increased both calcium-dependent (extra) ATPase activity and calcium-independent (basic) ATPase activity of vesicles. The apparent initial reaction rate of ATPase of $\text{n-}\text{alcohol-treated}$ vesicles was about twice that of control vesicles. With increasing number ($\text{n}$) of carbon atoms of the $\text{n-}\text{alcohols}$, the maximum increment of ATPase activity increased, and both the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$) required to inhibit calcium uptake by 50% and the alcohol concentration ($\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$) required to enhance ATPase activity by 50% of the maximum increment of ATPase activity decreased as follows.

$\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}\text{=23.5·10}{}^{\mathrm{?0.593n}}\text{M}$

$\text{N}{}_{\mathrm{ATPase}}\text{=35.5·10}{}^{\mathrm{?0.593n}}\text{M}$

The ratio, $\text{N}{}_{\mathrm{<mtext>ATPase</mtext>}}$ to $\text{N}{}_{\mathrm{<mtext>Ca</mtext>}}$, was constant for all $\text{n}$ values. The apparent free energy of binding of the methylene groups of $\text{n-}\text{alcohols}$ to sarcoplasmic reticulum vesicles was evaluated (?796 cal/mole) and compared with data from the partition of $\text{n-}\text{alcohols}$ in octanol and water (?670 cal/mole). The effects of $\text{n-}\text{alcohols}$ on membrane vesicles are discussed on the basis of these data.     

The energy barriers to ion transport by nonactin across thin lipid membranes

The experimental steady-state current-voltage relations for low concentrations of a neutral carrier and an ion may be fitted theoretically either by assuming a form for the potential dependence of the rate of transfer of complex across the membrane and adjusting the proposed nature of the association-dissociation reactions or by assuming equilibrium for the association and adjusting the potential dependence of the transfer process. Different dependences for the rate of transfer correspond, at least formally, to different shapes for the potential energy barrier which the complex must cross. By comparing measurements of the current-voltage relations for non-actin with Na⁺, K⁺, and NH₄⁺, it is possible to distinguish between the effeects of the various rates. For black lipid membranes made from glycerolmonooleate+$\text{n-}\text{hexadecane}$, the potential energy barrier is high with a narrow top, but the rate of association still becomes increasingly limiting for Na⁺, K⁺ and NH₄⁺, in the order given. For bacterial phosphatidylethanolamine, with $\text{n-}\text{decane}$ the barrier is much wider and no effect of the rate of association can be detected.     

A dynamic X-ray diffraction study of anesthesia action. Thickening of the myelin membrane by n-pentane

The structural changes induced in the myelin sheath by $\text{n-}\text{pentane}$ nerve impulse blockage were studied by small-angle X-ray diffraction using a linear position-sensitive detector. The results show that the thickness of the myelin period lattice increases from 170 to 180 Å during $\text{n-}\text{pentane}$ treatment.     

Side-specific analogues for the rat adipocyte sugar transport system

(1) 4,6-O-Ethylidene-d-glucose is a good inhibitor of adipocyte sugar transport from the external surface. Using radioactively labelled 4,6-O-methylidene-d-glucose we have shown that this compound is not taken up into cells by the hexose transporter but through a route which is insulin insensitive, d-glucose insensitive, temperature sensitive and which is slightly inhibited by phloretin. When efflux of 3-O-methyl-d-glucose is studied with 4,6-O-methylidene-d-glucose only present inside the cells then no detectable inhibition is observed indicating that this compound is a good side-specific analogue with a high affinity for only the external site of the hexose transporter. (2) Radioactively labelled alkyl-β-d-glucosides have been prepared. These also penetrate the adipocyte membrane by an insulin and d-glucose insensitive route. The half-times for equilibration with methyl-, n-propyl-, and $\text{n-}\text{butyl}\text{-β-}\text{d}\text{-}\text{glucosides}$ are 255, 9.45 and 3.3 min, respectively, indicating that the uptake rates are dependent upon the size of the alkyl group. (3) The glucosides show poor inhibition of 3-O-methyl-d-glucose transport when added to the external solution only. When cells are preincubated with $\text{n-}\text{propyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ and $\text{n-}\text{butyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ an increase in the amount of inhibition of 3-O-methyl-d-glucosez uptake is observed. This increase in inhibition correlates well with the glucoside uptake rates and indicates that availability of the glucosides at the internal surface of the transporter is required for inhibition. This has been confirmed by measuring 3-O-methyl-d-glucose exit in the presence of $\text{n-}\text{propyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ at the internal surface only. Thus, $\text{n-}\text{propyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ is a good side-specific analogue with high affinity only for the internal site of the hexose transporter. (4) $\text{n-}\text{Propyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ inhibition of d-allose transport is fully reversible. If cells are washed after a preincubation with the analogue then the inhibition is lost. The $\text{n-}\text{propyl}\text{-β-}\text{d}\text{-}\text{glucoside}$ inhibition of d-allose transport is reduced competitively by 3-O-methyl-d-glucose. (5) 6-O-Propyl-d-galactose has low affinity for both internal and external sites.     

Sensitivity of Na+-coupled d-glucose uptake,Mg2+-ATPase and sucrase to perturbations of the fluidity of brush-border membrane vesicles induced by n-aliphatic alcohols

The aim of our work is to show the importance of the role of hydrophobic bonds in maintaining Mg²⁺-ATPase or sucrase activity and Na⁺-coupled d-glucose uptake normal for the brush border of rat enterocytes. The activity of the two enzymes and the d-glucose uptake were therefore measured under the action of $\text{n-}\text{aliphatic alcohols}$ and related to the fluidity determined by ESR. Three concentrations were used for the first eight alcohols, those of octanol being about 1500-times lower than those of methanol. For each alcohol the d-glucose uptake and the fluidity were linear functions of the logarithm of the concentration, the linear regressions being practically parallel and equidistant. The concentrations ($\text{C}$) of the eight alcohols inhibiting the d-glucose uptake by 80% were similar to those increasing the membrane fluidity by 3%. The linear relationship which existed in both cases between log 1 / $\text{C}$ and log $\text{P, P}$ being octanol / water partition coefficients of the alcohols, was evidence of great sensitivity to the hydrophobic effect of the alcohols. Only the first alcohols, however, produced any notable inhibition of Mg²⁺-ATPase and sucrase. Hydrophobic bonds are thus shown to have little influence in maintaining the activity of Mg²⁺-ATPase and sucrase, but they modulate the Na⁺-coupled d-glucose uptake.     

Effects of gaseous anaesthetics and inert gases on the phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine

The phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine was studied under high pressures of helium (340 atm), nitrogen (340 atm), nitrous oxide (43 atm), cyclopropane (4.4 atm) and $\text{n-}\text{propane}$ (8.2 atm), using a turbidimetric technique. Helium and nitrogen increased the transition temperature by 0.021 and 0.006°C/atm, respectively, compared with 0.024°C/atm for hydrostatic pressure. Nitrous oxide reduced the transition by 0.58°C/atm. The hydrocarbon gases spread the transition width and lowered the transition temperature with increasing effect at higher doses. Comparisons with other membrane probes are made and the concentration of gases in the bilayer which lower the transition temperature by 1°C are estimated, in mol%: He, 10.2; N₂, 13.2; N₂O, 9.04; $\text{n-}\text{C}{}_3\text{H}{}_8$, 6.3 and cyclopropane, 12.8.