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W R Lieb

Publications and source records attributed to W R Lieb.

At least 55 records · Page 3Linked to original sources
PubMed1991

Role of hydrogen bonding in general anesthesia.

The importance of hydrogen bonding in determining the potency of a general anesthetic is controversial. In order to investigate the role of hydrogen bonding further, we have used a multiple linear regression approach to quantify the relative importance of various physical properties of an anesthetic molecule (i.e., its ability to donate or accept a hydrogen bond, its dipolarity and polarizability, and its size) in determining its anesthetic potency. For comparison, we have applied the same approach to partitioning between water and three simple, but contrasting solvents (n-octanol, n-hexadecane, and N,N-dimethylacetamide) and to inhibition of an enzyme (firefly luciferase) which mimics many of the properties of general anesthetic target sites in animals. We present equations which accurately predict potencies (over many orders of magnitude) for producing general anesthesia and inhibiting the firefly luciferase enzyme. We find that the aqueous potency (defined as the reciprocal of the aqueous EC50 concentration) of a molecule as a general anesthetic or an inhibitor of luciferase is determined overwhelmingly by its size (which increases potency) and its ability to accept a hydrogen bond (which decreases potency), but only marginally by its ability to donate a hydrogen bond or by its dipolarity and polarizability. We conclude that general anesthetic target sites in animals must have, in addition to their overall hydrophobicity, a polar component which is a relatively poor hydrogen bond donor, but which can accept a hydrogen bond about as well as water.

Adenosine Triphosphate↗
PubMed1991

Effects of physiologically relevant pressures of helium on the structure of cholesterol-containing lipid bilayers. A neutron diffraction study.

We have used neutron diffraction to study the effects of helium gas (1-210 atm) on the structure of a lipid bilayer model of neuronal plasma membranes. We have recorded diffraction patterns from hydrated multilayers of dimyristoyl lecithin and 40% (molar) cholesterol to a resolution of approximately 6.5 A and have calculated scattering amplitude density distributions as a function of pressure. We find that there are no significant changes in the scattering density profiles at 95% confidence over the range of pressures investigated, suggesting that the physiological effects of high helium pressure are unlikely to be a consequence of changes in the structures of the lipid bilayer portions of membranes.

Biophysical Phenomena↗
PubMed1991

Anesthetic inhibition of firefly luciferase, a protein model for general anesthesia, does not exhibit pressure reversal.

The surprising observation that pressures of the order of 150 atmospheres can restore consciousness to an anesthetized animal has long been central to theories of the molecular mechanisms underlying general anesthesia. We have constructed a high-pressure gas chamber to test for "pressure reversal" of the best available protein model of general anesthetic target sites: the pure enzyme firefly luciferase, which accounts extremely well for animal potencies (over a 100,000-fold range). We found no significant pressure reversal for a variety of anesthetics of differing size and polarity. It thus appears that either firefly luciferase is not an adequate model for general anesthetic target sites or that pressure and anesthetics act at different molecular sites in the central nervous system.

1-Octanol↗
PubMed1991

Probing the molecular dimensions of general anaesthetic target sites in tadpoles (Xenopus laevis) and model systems using cycloalcohols.

1. The series of cycloalcohols C6, C7, C8 and C10 have been used to probe the molecular dimensions of a variety of general anaesthetic target sites. 2. The general anaesthetic EC50 concentrations of the cycloalcohols were determined for tadpoles (Xenopus laevis). All of the cycloalcohols tested were found to be potent general anaesthetics (on average EC50/Csat = 0.03). 3. The effects of the cycloalcohols on highly purified luciferase enzymes from fireflies (Photinus pyralis) and bacteria (Vibrio harveyi) were also investigated. Both enzymes were inhibited competitively, with the cycloalcohols competing with firefly luciferin for binding to the firefly enzyme and with n-decanal for binding to the bacterial enzyme. 4. The binding site on the firefly enzyme could accommodate two molecules of cycloalcohols C6 and C7 but only a single molecule of the larger cycloalcohols (C8 and C10), implying a volume of the binding site of about 250 cm3 mol-1. In contrast, the binding site on the bacterial luciferase could bind only a single cycloalcohol molecule between C6 and C10. 5. While all of the cycloalcohols were potent inhibitors of the firefly luciferase enzyme (on average EC50/Csat = 0.015), they were very weak inhibitors of the bacterial luciferase enzyme (on average EC50/Csat = 0.12). Since both enzymes bind long-chain aliphatic n-alcohols tightly, the differing affinities of the cycloalcohols for the two enzymes is probably a consequence of geometrical factors. 6. The cycloalcohols produced very small effects on lipid bilayers. At EC50 concentrations which produce general anaesthesia, lipid bilayer phase transitions were shifted, on average, by only 0.43 degrees C. 7. We conclude that the general anaesthetic effects of the cycloalcohols can most economically be explained by assuming that the cycloalcohols act at protein binding sites in the central nervous system. These target sites would have binding properties similar to those of the anaesthetic-binding site on firefly luciferase, but their average volume would be somewhat smaller than 250 cm3 mol -1.

Alcohols↗
PubMed1991

An anaesthetic-activated potassium channel.

We have discovered a novel potassium current in an identified molluscan neuron which is activated at surgical levels of volatile general anaesthetics. The current is not significantly voltage-gated and persists for as long as the anaesthetic is present. The anaesthetic response is completely reversibly and saturates at low anaesthetic partial pressures. The current is absent in surrounding neurons.

Animals↗
PubMed1990

Effects of general anesthetics on the bacterial luciferase enzyme from Vibrio harveyi: an anesthetic target site with differential sensitivity.

The effects of a diverse range of 36 general anesthetics and anesthetic-like compounds on a highly purified preparation of the bacterial luciferase enzyme from Vibrio harveyi have been investigated. Under conditions where the flavin site was saturated, almost all of the anesthetics inhibited the peak enzyme activity and slowed the rate of decay. However, a small number of the more polar agents only inhibited at high concentrations, while stimulating activity at lower concentrations. The inhibition was found to be competitive in nature, with the anesthetics acting by competing for the binding of the aldehyde substrate n-decanal. The anesthetic binding site on the enzyme could accommodate only a single molecule of a large anesthetic but more than one molecule of a small anesthetic, consistent with the site having circumscribed dimensions. The homologous series of n-alcohols and n-alkanes exhibited cutoffs in inhibitory potency, but these cutoffs occurred at very different chain lengths (about C10 for the n-alkanes and C15 for the n-alcohols), mimicking similar cutoffs observed for general anesthetic potencies in animals. Binding constants determined from peak height measurements showed that the inhibitor binding site was predominantly hydrophobic (with a mean delta delta G CH2 of -5.0 kJ/mol), but fluctuations in the binding constants with chain length revealed regions in the binding site with polar characteristics. Binding constants to an intermediate form of the enzyme (intermediate II) were also determined, and these confirmed the principal features of the binding site deduced from the peak height measurements. The long-chain compounds, however, bound considerably tighter to the intermediate II form of the enzyme, and this was shown to account for the biphasic decay kinetics that were observed with these compounds. Overall, there was poor agreement between the EC50 concentrations for inhibiting the luciferase enzyme from V. harveyi and those which induce general anesthesia in animals, with bulky compounds being much less potent, and moderately long chain alcohols being much more potent, as luciferase inhibitors than as general anesthetics.

Alcohols↗
PubMed1990

Mechanisms of general anesthesia.

Although general anesthetics are often said to be nonspecific agents, it is likely that they act at a much more restricted set of target sites than commonly believed. The traditional view has been that the primary targets are lipid portions of nerve membranes, but recent evidence shows that the effects on lipid bilayers of clinically relevant levels of anesthetics are very small. Effects on most proteins are also small, but there are notable examples of proteins that are extremely sensitive to anesthetics and mimic the pharmacological profile of anesthetic target sites in animals. Such target sites are amphiphilic in nature, having both hydrophobic and polar components. The polar components appear to behave as good hydrogen-bond acceptors but poor hydrogen-bond donors. Although the targets can accept molecules with a wide variety of shapes and chemical groupings, they are unaffected by molecules exceeding a certain size. Overall, the data can be explained by supposing that the primary target sites underlying general anesthesia are amphiphilic pockets of circumscribed dimensions on particularly sensitive proteins in the central nervous system.

Anesthesia, General↗
PubMed1988

Volatile general anaesthetics activate a novel neuronal K+ current.

Although it is still controversial whether the primary target sites underlying general anaesthesia are proteins or lipids, it is generally thought that the ultimate targets are ion channels in nerve membranes. One approach to finding these targets is to study the effects of general anaesthetics on identified neurons, where differential effects on neuronal activity can be pursued to the molecular level. Here we report that amongst a group of apparently identical molluscan neurons having endogenous firing activity, a single cell displays an unusual sensitivity to volatile agents (which, at surgical levels, completely inhibit its activity). We further show that this sensitivity is due to a novel anaesthetic-activated K+ current, which is found in the sensitive cell but not in the surrounding insensitive cells. This K+ conductance is not appreciably voltage-gated and persists for as long as the anaesthetic is present. The response to anaesthetics is completely reversible and saturates at low anaesthetic partial pressures: the half-maximal response for halothane occurs at 0.0063 atm, close to its minimum alveolar concentration (0.0075 atm) in man.

Animals↗
PubMed1987

Are the biological effects of ethanol due to primary interactions with lipids or with proteins?

The biological effects of ethanol and other simple organic molecules have traditionally been ascribed to their actions on the lipid portions of biological membranes. However, at the concentrations at which these molecules exert their pharmacological effects in man and other animals, changes in the structure and fluidity of lipid bilayers are extremely small and can be mimicked by a change in temperature of only about 1 degree C. On the other hand, we have found that the activity of a soluble protein can be markedly affected at these concentrations. Indeed, the concentrations of general anaesthetics which half-inhibit the activity of this enzyme are essentially identical to those which cause general anaesthesia in animals. The evidence thus suggests that ethanol and other simple molecules act directly on proteins rather than lipids.

Anesthetics↗
PubMed1986

The pharmacology of simple molecules.

The biological effects of simple molecules have traditionally been ascribed to their actions on the lipid portions of biological membranes. However, at the low concentrations of these molecules which induce general anaesthesia or have acute toxic effects in animals, changes in lipid bilayer properties are so small that they are unlikely to be relevant biologically. On the other hand, these molecules do inhibit the activity of a pure lipid-free protein, with ED50 concentrations which are essentially identical to the biological ED50 and LD50 concentrations. Moreover, the well-known but puzzling cutoffs in potencies in homologous series of compounds are also found with this enzyme. The accumulating evidence now suggests that the pharmacological effects of low concentrations of relatively inert agents are best explained in terms of their direct binding to amphiphilic pockets of circumscribed dimensions on proteins.

Anesthetics↗
PubMed1986

Partitioning of long-chain alcohols into lipid bilayers: implications for mechanisms of general anesthesia.

Alcohols act as anesthetics only up to a certain chain length, beyond which their biological activity disappears. Although the molecular nature of general anesthetic target sites remains unknown, presently available data support the hypothesis that this "cutoff" in anesthetic activity could be due to a corresponding cutoff in the absorption of long-chain alcohols into lipid-bilayer portions of nerve membranes. To test this hypothesis, we have developed an extremely sensitive biological assay, based on inhibition of the light-emitting firefly luciferase reaction, which is capable of measuring lipid-bilayer/buffer partition coefficients K for very lipid soluble compounds. Contrary to the hypothesis and reported data, we find a strictly linear increase in log(K) as the chain length increases [delta(delta G0)CH2 = - 3.63 kJ/mol] for the primary alcohols from decanol to pentadecanol, with no hint of a cutoff. The fact that alcohols continue to partition into lipid bilayers long after their biological activity has ceased is consistent with the view that the primary target sites in general anesthesia are proteins rather than the lipid-bilayer portions of nerve membranes.

Absorption↗
PubMed1982

Molecular mechanisms of general anaesthesia.

Important constraints on possible molecular mechanisms of general anaesthesia are derived from a quantitative reappraisal of data on the potency of general anaesthetics on whole animals. Despite their popularity, theories that invoke lipids as the prime target do not look at all promising, and available data point much more plausibly to a direct effect on particularly sensitive proteins. Structural changes of proteins on binding general anaesthetics are probably small but may be sufficient to perturb normal function; alternatively, anaesthetics may compete with an endogenous ligand. The phenomenon of pressure reversal of anaesthesia may simply be due to anaesthetics being squeezed away from their target sites.

Anesthesia, General↗
PubMed1982

Do clinical levels of general anaesthetics affect lipid bilayers? Evidence from Raman scattering.

We have used Raman spectroscopy to investigate the effects of the general anaesthetics halothane and chloroform on lipid bilayer order. Clinical concentrations of these anaesthetics had no significant effect on the hydrocarbon chain conformation in multilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol. This result was obtained with a technique sufficiently precise to monitor changes in the acyl chain trans-gauche population ratio associated with a 1-2 K alteration in temperature. Very high levels of anaesthetics caused a marked disordering of the hydrocarbon chains. The danger of inferring an effect at clinical concentrations from data obtained at much higher levels is illustrated by a statistical analysis of our dose-response curves.

Cholesterol↗
PubMed1982

Combined effects of ATP and phosphate on rubidium exchange mediated by Na-K-ATPase reconstituted into phospholipid vesicles.

1. Phospholipid vesicles reconstituted with Na-K-ATPase show an (ATP+phosphate)-stimulated Rb-Rb exchange, with properties similar to the K-K exchange of human red cells. This includes a rate 15-20% of the rate of active ATP-dependent Na-K exchange.2. We have studied activation of this Rb-Rb exchange by ATP at fixed phosphate concentrations and by phosphate at fixed ATP concentrations. It is found for both ATP and phosphate that with low concentrations of the fixed ligand an increase in concentration of the complementary ligand produces first stimulation and then inhibition of Rb-Rb exchange. At high concentrations of the fixed ligand the complementary ligand shows only saturation behaviour.3. The pattern of activation and of inhibition by ATP and by phosphate is affected by the Rb(0) concentration in the exterior medium, in that higher concentrations of Rb(0) counteract inhibitory effects of high concentrations of ATP and phosphate.4. (ATP+phosphate)-stimulated Rb-Rb exchange is activated by Rb(0) in the exterior medium along a sigmoid curve. An increase of Rb(i) within the vesicles, which raises the maximal velocity of Rb-Rb exchange, is accompanied by a smaller increase in the Rb(0) concentration required for half-maximal stimulation of the Rb-Rb exchange.5. The data are interpreted in terms of a model similar to those proposed by Karlish & Stein (1982a,b), but extended to include simultaneous effects of ATP and phosphate. Inhibitions by high concentration of ATP or phosphate arise as a result of stabilization of E(1) ATP or E(2)-P forms respectively, in the presence of low concentrations of the complementary ligand. With high concentrations of the fixed ligand, saturation behaviour of the varying ligand is observed because the occluded Rb forms become the dominant transport intermediates. The occluded Rb forms bind both ATP and phosphate weakly and independently. The effects of ATP together with phosphate are accounted for by a simple combination of their separate effects on the Rb-Rb exchange.6. We suggest that the functional role of the occluded Rb form E(2) (Rb)(occ) in active transport is to minimize passive cation leaks through the system and allow control of the direction of cation movements by binding of physiological ligands such as ATP or phosphate.

Adenosine Triphosphate↗
PubMed1981

Is membrane expansion relevant to anaesthesia?

General anaesthesia can be induced by a wide variety of structurally dissimilar molecules. Consequently, the mechanism must involve some rather nonspecific interactions as the target site, generally held to be in nerve membranes. The primary site of action has been postulated to be either lipid or protein or both. Although recent work has cast doubt on the lipid hypotheses, protein models still flourish. In particular, Seeman and his co-workers have shown that biological membranes expand when anaesthetic molecules are added, and that this expansion is far greater than that which occurs with lipid bilayers. It has been suggested that this difference is due to extensive conformational changes in the membrane proteins, and several mechanisms have been proposed to explain this large expansion of proteins. We now report the first direct measurements of the volumes occupied by general anaesthetic molecules in both biological membranes and lipid bilayers. We show that, in fact, biological membranes expand much less and lipid bilayers expand more than previously reported. The volume that a general anaesthetic molecule occupies is essentially the same in biological membranes, lipid bilayers and water. Our results lead us to question all generalized membrane expansion hypotheses for the mechanism of general anaesthesia, in favour of hypotheses would include more specialized target sites.

Anesthesia, General↗