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N P Franks

Publications and source records attributed to N P Franks.

At least 19 recordsLinked to original sources
PubMed1995

Actions of general anaesthetics on a neuronal nicotinic acetylcholine receptor in isolated identified neurones of Lymnaea stagnalis.

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were studied under two-electrode voltage-clamp. Neuronal nicotinic acetylcholine receptor currents were studied at low acetylcholine (ACh) concentrations (< or = 200 nM). At these levels, control currents were non-desensitizing and proportional to the square of the ACh concentration. 2. IC50 concentrations were determined for the steady-state inhibition of the ACh-activated current by 31 general anaesthetics plus the non-anaesthetic alcohol n-tridecanol. The general anaesthetics included inhalational agents, n-alcohols, n-alkane-(alpha,omega)-diols, cycloalcohols and an n-alkane. 3. Anaesthetic inhibition was independent of voltage and consistent with two anaesthetic-binding sites on the receptor. 4. IC50 concentrations for inhibiting the neuronal nicotinic ACh receptor correlated well (r = 0.97) with EC50 concentrations for general anaesthesia. The maximum deviation from the line of identity was less than fourfold. The inhalational agents tended to be more potent as inhibitors of the ACh receptor than as general anaesthetics, while the alcohols and diols were less potent. 5. The inhibition of the ACh-induced current by the homologous series of n-alcohols exhibited a cutoff at the same position (just after dodecanol) as found for the induction of general anaesthesia in tadpoles. 6. Polarity profile maps of the anaesthetic-binding sites on the neuronal nicotinic ACh receptor were calculated from IC50 concentrations for the homologous series of n-alcohols and n-alkane-(alpha,omega)-diols. They reveal amphiphilic sites with apolar regions capable of accommodating the hydrocarbon chains of n-alcohols as large as decanol. A striking resemblance was found to profiles previously calculated from data for tadpole general anaesthesia.

Acetylcholine↗
PubMed1994

Molecular and cellular mechanisms of general anaesthesia.

General anaesthetics are much more selective than is usually appreciated and may act by binding to only a small number of targets in the central nervous system. At surgical concentrations their principal effects are on ligand-gated (rather than voltage-gated) ion channels, with potentiation of postsynaptic inhibitory channel activity best fitting the pharmacological profile observed in general anaesthesia. Although the role of second messengers remains uncertain, it is now clear that anaesthetics act directly on proteins rather than on lipids.

Anesthesia, General↗
PubMed1994

Can the stereoselective effects of the anesthetic isoflurane be accounted for by lipid solubility?

Isoflurane is an inhalational general anesthetic widely used in surgical operations as a racemic mixture of its two optical isomers. The recent availability of pure enantiomers of isoflurane has encouraged their use in experimental studies, and stereoselective effects have now been observed on anesthetic-sensitive neuronal ion channels. Although it has been assumed that such chiral effects demonstrate direct interactions with proteins, it is possible that they could be due to stereoselective interactions with chiral membrane lipids. We have determined the partition coefficients of the two optical isomers of isoflurane between lipid bilayers and water, using racemic isoflurane and gas chromatography with a chiral column. For lipid bilayers of phosphatidylcholine (PC) and 4 mol% phosphatidic acid (PA), both with and without cholesterol (CHOL), we found equal partitioning of the isoflurane optical isomers. The ratios of the S(+) to R(-) isoflurane partition coefficients were (mean +/- SEM): 1.018 +/- 0.010 for bilayers of PC/CHOL/PA (mole ratios 56:40:4) and 1.011 +/- 0.002 for bilayers of PC/PA (mole ratio 96:4). Molar partition coefficients for racemic isoflurane were 49 +/- 4 and 165 +/- 10, respectively. These findings support the view that the stereoselective effects on ion channels observed with isoflurane are due to direct actions on proteins rather than lipids.

Animals↗
PubMed1994

Insensitivity of P-type calcium channels to inhalational and intravenous general anesthetics.

BACKGROUND: Voltage-gated Ca2+ channels long have been considered plausible targets for general anesthetics. Previous anesthetic studies have focused on L-, T-, or N-type channels, but there have been no studies on channels identified as P-type. Since P-type channels may be the most important voltage-gated Ca2+ channels involved in synaptic transmission in mammalian brain, it is important to establish their sensitivity to clinically relevant concentrations of general anesthetics. METHODS: Acutely dissociated cerebellar Purkinje neurons were obtained from 7-14-day-old Sprague-Dawley rats. P-type currents were measured using the whole-cell version of the patch-clamp technique, with Ba2+ as the current carrier. General anesthetics were applied to the neurons in aqueous solution at room temperature (20-23 degrees C). RESULTS: P-type Ca2+ channels were found to be very insensitive to a variety of general anesthetics and ethanol. Inhibitions of less than 10% were produced by 0.35 mM halothane, 0.35 mM isoflurane, 32 microM thiopental, 50 microM pentobarbital, 2 microM propofol, and 200 mM ethanol. Substantial anesthetic inhibition was found only at free aqueous concentrations much greater than those that are clinically relevant. For halothane, the dose-response curve showed an IC50 concentration of 1.17 +/- 0.02 mM and a Hill coefficient of 2.02 +/- 0.04 (mean +/- SEM). CONCLUSIONS: The relatively small inhibitions of P-type Ca2+ channels produced by volatile and intravenous anesthetics at their free aqueous EC50 concentrations for general anesthesia in mammals suggest that these channels do not play a major role in the induction of general anesthesia.

Administration, Inhalation↗
PubMed1994

Stereoselective and non-stereoselective actions of isoflurane on the GABAA receptor.

1. Acutely dissociated cerebellar Purkinje neurones from 8-14 day old rats were studied under voltage clamp in the whole-cell patch-clamp configuration. Cl- currents induced by bath application of gamma-aminobutyric acid (GABA) were measured (using symmetrical Cl- solutions) at both low (2 microM) non-desensitizing and high (300 microM) desensitizing concentrations of GABA. 2. At 2 microM GABA, the bicuculline-sensitive Cl- currents were potentiated by racemic isoflurane and both of its optical isomers. Isoflurane had no effect on membrane current in the absence of GABA. The dose-response data for potentiation by racemic isoflurane could be fitted with a Hill equation with an EC50 = 320 +/- 20 microM isoflurane and a Hill coefficient of h = 2.7 +/- 0.4 (means +/- s.e.mean). 3. The potentiations produced by the optical isomers of isoflurane at 2 microM GABA were stereoselective at moderate and high anaesthetic concentrations. The maximum stereoselectivity, about two fold, occurred at the EC50 concentration for general anaesthesia (310 microM isoflurane), with S(+)-isoflurane being more effective than R(-)-isoflurane. At sub-anaesthetic concentrations, the stereoselectivity was less marked and vanished at the lowest concentration used (77 microM isoflurane). 4. The sustained residual current remaining after exposure of neurons to a desensitizing concentration of GABA (300 microM) was inhibited non-stereoselectively, but only at high concentrations of isoflurane. The ratio of inhibitions by S(+)- and R(-)-isoflurane (mean +/- s.e.mean) was 1.14 +/- 0.21 at 770 microM isoflurane. At the EC50 concentration for general anaesthesia, however, the inhibition was barely significant. 5. The above results are discussed in relation to the possible role of the GABAA receptor channel in general anaesthesia.

Anesthesia, General↗
PubMed1993

A simple method for recording single-channel activity from synaptic plasma membranes.

Due to the small size of most nerve terminals, the ion channels which underlie presynaptic currents are usually inaccessible to investigation by conventional electrophysiological techniques. Here we describe a simple method for obtaining single-channel recordings from synaptic plasma membranes that does not require exposure of the native membranes to exogenous lipids or fusogens. To illustrate the method, we have recorded single-channel activity from rat cerebrocortical synaptosomal membranes. Under conditions designed to isolate calcium-independent currents, we describe three channel types that are most commonly observed.

Action Potentials↗
PubMed1992

Effects of temperature on the anaesthetic potency of halothane, enflurane and ethanol in Daphnia magna (Cladocera: Crustacea).

1. The effects of temperature on the anesthetic potencies of halothane, enflurane and ethanol have been studied in the water flea Daphnia magna. 2. In the absence of anaesthetics, decreasing temperature resulted in decreased activity by the daphnids. 3. Potencies in the gas phase decreased with increasing temperature for all of the anaesthetics, while aqueous potency decreased for halothane and enflurane but increased for ethanol. 4. Enthalpy calculations suggest that the observed potency changes for the inhalational anaesthetics cannot be accounted for in terms of changing solubility in lipid bilayers but most likely reflect more specific interactions with animal target sites.

Animals↗
PubMed1991

Mapping the polarity profiles of general anesthetic target sites using n-alkane-(alpha, omega)-diols.

The effects of the homologous series of n-alkane-(alpha, omega)-diols have been studied on the inhibition of the purified firefly luciferase enzyme from Photinus pyralis, the inhibition of the purified bacterial luciferase enzyme from Vibrio harveyi, and the induction of general anesthesia in Xenopus laevis tadpoles. All but one of the diols tested were found to be reversible general anesthetics. The diols inhibited firefly luciferase by competing with its normal substrate firefly luciferin, and they inhibited bacterial luciferase by competing with the substrate n-decanal. For all but the smallest agent (1,4-butanediol), only a single diol molecule was found to be involved in the inhibition of the enzymes. Inhibition constants Ki were determined for the enzymes, and general anesthetic EC50 concentrations were determined for tadpoles. These data were then used in conjunction with previously determined n-alkane and n-alcohol data to calculate, as a function of chain length, the incremental standard Gibbs free energies delta (delta G0) for adding apolar -CH2- groups and for converting apolar terminal -CH3 groups to polar -CH2OH groups. The resulting plots of delta (delta G0) versus chain length gave a consistent mapping of the polarity profiles of the anesthetic-binding pockets. They clearly reveal the existence of two substantial and distinct polar regions in the anesthetic-binding pocket of firefly luciferase but only one such region for bacterial luciferase and for the unknown target sites underlying general anesthesia. The polarities and geometric properties of these different binding sites for straight-chain anesthetics are discussed in terms of simple models.

Anesthetics↗
PubMed1991

Stereospecific effects of inhalational general anesthetic optical isomers on nerve ion channels.

Although it is generally agreed that general anesthetics ultimately act on neuronal ion channels, there is considerable controversy over whether this occurs by direct binding to protein or secondarily by nonspecific perturbation of lipids. Very pure optical isomers of the inhalational general anesthetic isoflurane exhibited clear stereoselectivity in their effects on particularly sensitive ion channels in identified molluscan central nervous system neurons. At the human median effect dose (ED50) for general anesthesia, the (+)-isomer was about twofold more effective than the (-)-isomer both in eliciting the anesthetic-activated potassium current IK(An) and in inhibiting a current mediated by neuronal nicotinic acetylcholine receptors. For inhibiting the much less sensitive transient potassium current IA, the (-)-isomer was marginally more potent than the (+)-isomer. Both isomers were equally effective at disrupting lipid bilayers.

Anesthesia, Inhalation↗
PubMed1991

Modulation of the general anesthetic sensitivity of a protein: a transition between two forms of firefly luciferase.

The activities of most proteins are relatively insensitive to general anesthetics. A notable exception is firefly luciferase, whose sensitivity to a wide range of anesthetic agents closely parallels that of whole animals. We have now found that this sensitivity can be controlled by ATP. The enzyme is insensitive at low (microM) concentrations of ATP and very sensitive at high (mM) concentrations. The differential sensitivity varies from anesthetic to anesthetic, being greatest (about a 100-fold difference) for molecules with large apolar segments. This suggests that anesthetic sensitivity is modulated by changes in the hydrophobicity of the anesthetic-binding pocket. Parallel changes in the binding of the substrate firefly luciferin, for which anesthetics compete, indicate that anesthetics bind at the same site as the luciferin substrate. These changes in the nature of the binding pocket modify not only the sensitivity to anesthetics but also the position of the "cutoff" in the homologous series of primary alcohol anesthetics; the cutoff position can vary from octanol to pentadecanol, depending upon the concentration of ATP. Our results suggest that particularly sensitive anesthetic target sites in the central nervous system may possess anesthetic-binding pockets whose polarities are regulated by neuromodulatory agents.

Adenosine Triphosphate↗
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↗